Types Of Cl Research Articles

Three distinct chloride channels control anion movements in rat parotid acinar cells.

1. We used the whole-cell configuration of the patch clamp technique to examine the different macroscopic Cl- currents present in single rat parotid acinar cells. 2. Cell swelling produced by negative osmotic pressure (hypotonic bath solutions) induced a large outwardly rectifying Cl- current with little or no time and voltage dependence. In contrast, an increase in intracellular [Ca2+] induced by ionomycin activated Cl- currents with very different properties. Ca(2+)-activated Cl- currents showed outward rectification, relatively slow activation kinetics and marked voltage dependence. These results are consistent with the existence of two different outwardly rectifying Cl- channels in rat parotid cells. 3. In conditions designed to eliminate the activation of these two Cl- currents, a third type of current was observed. This third current was activated in a time-dependent manner by hyperpolarized potentials and was about equally permeant to Cl-, I- and Br-. 4. The properties of the hyperpolarization-activated current were similar to those of the cloned ClC-2 channel. Polymerase chain reaction-based methods and ribonuclease protection analyses indicated the presence in parotid gland of mRNA homologous to ClC-2. 5. Individual parotid acinar cells expressed all three types of Cl- channels. Each type of channel may contribute to Cl- efflux in distinct stages of the secretion process depending on the intracellular [Ca2+], cell volume and membrane potential.

Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor.

Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membrane depolarization by activating a new type of Cl- channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers.

Hormonal regulation of cardiac cystic fibrosis transmembrane conductance regulator chloride channels.

The chloride conductance that arises from the stimulation of beta-adrenoceptors has been shown to be carried by a cardiac isoform of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. This brief review will focus first upon the cellular signal transduction system for the activation of this type of Cl- channels and then its regulation by catecholamines, muscarinic agonists, endothelin-1 and angiotensin-II. Both in physiological and pathological conditions, the complex interaction of these agonists modulates the Cl- conductance, which is potentially arrhythmogenic by shortening the action potential duration and inducing the depolarization.

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

1. The patch-clamp technique was used to characterize chloride channels from the apical membranes of bovine tracheal epithelial cells. Application of GTP gamma S or NaF to excised patches revealed the existence of a novel type of Cl- channel regulated by G-proteins in a membrane-delimited manner. 2. The channel had a linear current-voltage relationship, with a conductance of 100-120 pS. Its open probability was independent of voltage. 3. The channel was highly anion selective (permeability ratio, PNa/PCl = 0.06 +/- 0.04) and had the halide permeability sequence: I- > Br- > or = Cl- > F-, corresponding to the Eisenman I sequence. This suggested that neither ionic size nor diffusion rate determined ion permeation through the channel. 4. The mole fraction behaviour was studied using fluoride and chloride ions. Mixtures of ions produced currents that would be expected from the linear combination of the two ions acting independently, indicating relatively simple permeation through the pore and compatible with a single ion binding site. 5. The channel was inhibited by the stilbene disulphonates SITS (4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulphonic acid) and DNDS (4,4'-dinitrostilbene-2,2'-sulphonic acid). SITS introduced voltage dependence to channel gating and indicated the possible involvement of lysine residues in the channel permeation pathway. 6. NaF was unable to activate Cl- channels in the presence of the aluminum chelator, deferoxamine mesylate. This indicates that Al3+ ions play an important role in chloride channel activation by fluoride. NaF activation was not dependent on the presence of calcium ions. 7. The channel was insensitive to alkaline phosphatase and to the specific inhibitors of protein phosphatase types I and 2A, okadaic acid and calyculin A. 8. The channels could be activated by GTP gamma S or by NaF in the presence of the phospholipase A2 inhibitor quinacrine, indicating that this enzyme is not involved in channel regulation.

Regulatory volume decrease in a renal distal tubular cell line (A6) I. Role of K+ and Cl−

Changes in volume of A6 epithelial cells were monitored by recording cell thickness (Tc). The response of Tc to a reduction of the basolateral osmolality from 260 to 140 mosmol/kg was recorded while transepithelial Na+ transport was inhibited by 20 microM amiloride. With Cl--containing bathing media, this osmotic challenge elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). Substitution of SO4(2-) or gluconate for Cl- markedly reduced the RVD, whereas cells completely maintained their ability to regulate their volume after replacing Cl- by NO3(-). A conductive pathway for Cl- excretion is suggested, which is insensitive to NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid], an inhibitor of some types of Cl- channels. Ba2+ (5 or 20 mM) reduced the RVD. A more pronounced inhibition of the RVD was obtained with 500 microM quinine, a potent blocker of volume-activated K+ channels. K+-induced depolarization of the basolateral membranes of tissues incubated with SO4(2-)-containing solutions completely abolished the RVD. Noise analysis in the presence of Ba2+ showed the activation of an apical K+ conductive pathway. These results demonstrate that cell volume regulation is controlled by processes involving Cl- and K+ excretion through conductive pathways.

Extracellular ATP activates both Ca2+- and cAMP-dependent Cl? conductances in rat epididymal cells

Activation of Ca2+ and cAMP-dependent Cl- conductances by extracellular ATP was studied using the whole-cell patch clamp technique. Immediately after addition of extracellular ATP (10 microM), activation of whole-cell Cl- current exhibiting delayed inactivation and activation kinetics at hyperpolarizing and depolarizing voltages, respectively, was observed. After prolonged activation, the kinetic characteristics of the ATP-induced Cl- current became time- and voltage-independent. When applied to the later phase of the ATP-activated whole-cell current, the disulfonic acid stilbene DIDS (200 microM) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mM) completely inhibited it. Inclusion of a peptide inhibitor for protein kinase A (PKI, 10 nM) in the pipette solution blocked ATP-induced time- and voltage-independent current activation but did not affect the delayed activating and inactivating current activation but did not affect the delayed activating and inactivating current which could be totally blocked by DIDS. Anion selectivity sequence was determined in the presence of either PKI or DIDS and found to be significantly different. Increased pipette EGTA (10 mM) or treatment of the cells with trifluoperazine (40 microM), an inhibitor of calmodulin, suppressed both types of ATP-induced Cl- currents. No current activation by ATP was observed when cells were dialyzed with the IP3 receptor blocker, heparin (10 ng/ml). These results suggest that extracellular ATP activates IP3-linked Ca(2+)-dependent regulatory pathway, which in turn activates cAMP-dependent pathway, leading to activation of both Ca2+ and cAMP-dependent Cl- conductances in epididymal cells.

Cl? channel inhibition by glibenclamide is not specific for the CFTR-type Cl? channel

As long as the question of which channels are responsible for cAMP-mediated epithelial Cl- secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl- currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl- channels of HT29 and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low-conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl- channel which, potentially, may contribute to transepithelial Cl- secretion.

Prostaglandins and sex steroids from corpora lutea of pregnant mares. In vitro studies

To delineate endocrine mechanisms regulating equine luteal function and the possible functional differences between one month and three month corpora lutea of pregnancy (CL), the in vitro basal releases of prostaglandin F (PGF), prostaglandin E 2 (PGE 2), progesterone, and 17β-estradiol by one and three month CL of pregnant mares were evaluated. In addition, the in vitro effects of PGF 2 β PGE 2, progesterone and 17β-estradiol on synthesis of prostaglandins (PGs) and sex steroids were studied. PGF, PGE 2, and 17β-estradiol secretion was higher in one month than in three month CL, while progesterone was secreted similarly by both types of CL. PGE 2 treatment decreased 17β-estradiol in one month CL; progesterone increased PGE 2 in one and three month CL; 17β-estradiol increased PGE 2 in one month CL. This study suggests that, in one month CL, PGE 2 production could be regulated by progesterone and 17β-estradiol, while, in three month CL, this prostaglandin seems to be under the control of progesterone alone.

Cl? and K+ channel currents during the action potential in Chara. simultaneous recording of membrane voltage and patch currents

Patch currents in the cell attached-mode and action potentials (AP) have been recorded simultaneously in internodal cells of Chara corallina. The action potentials are closely correlated with transient patch currents. With pipettes containing either 50 mM CaCl2 or 100 mM KCl plus 1 or 5 mM CaCl2, these transients measured up to 100 to 200 pA per patch at zero mV. Transients had a mean duration (time during which the current was > or = half maximum peak amplitude) of about 1 sec, a maximum slope for current rising of about 400 pA sec-1 and a maximum rate of about 100 pA sec-1 for current decay, with no obvious effect of external Ca2+ on either of these parameters. In well-resolved recordings of current transients triggered by an action potential (AP), activities of two types of Cl--conducting channels (15 and 38 pS) have been identified. Since activity of these channels was only observed during action potentials but not upon positive voltage steps, these channels are not directly voltage gated but point to a cytoplasmic gating factor which accumulates during excitation and propagates from excited areas to the patch. A K(+)-conducting channel (40 pS) could be identified as well during an AP, when 100 mM KCl was in the pipette solution. The activity of this channel relaxed at the end of the APs with a time constant of about 3 sec. Stimulated activity of this channel is understood to cause the repolarization overshoot during the final phase of the action potential, whereas the transient activation of the Cl- channels determines the fast voltage changes of the action potential.

A chloride channel reconstituted from fetal rat brain growth cones.

Chloride channels were reconstituted into planar lipid bilayers isolated from a preparation of growth cone particles (GCPs) isolated from fetal rat brain. One type of channel was predominantly seen and some of its biophysical and pharmacological properties were studied. The single channel i-V relationship was curvilinear with a chord conductance of 75 pS at +30 mV in symmetric 200 mM NaCl solutions buffered with phosphate. The channel was inactivated by depolarization, and this inactivation was reversed rapidly upon returning to -25 mV. The Cl- channel was significantly permeant to Na+ ions (PNa/PCl = 0.26), and the relative halide permeabilities were determined to be: I(1.92) > Br(1.73) > Cl(1.0) > F(0.34). The channel was inhibited by the common stilbene compounds (DIDS, SITS, DNDS), as well as by Zn2+ ions and an indanyloxyacetic acid derivative. A developmental role for the GCP Cl- channel is suggested by the observation that adult rat brain synaptosomal membranes were nearly devoid of this type of Cl- channel.

CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance.

Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.

Chloride channels in apical membrane of primary cultures of rabbit distal bright convoluted tubule

Using the patch clamp technique on the apical membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS was observed in 9% of the patches. Cells pretreated with 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or 5 microM forskolin increased the expression of Cl- channels by 26 and 37%, respectively. In cell-attached and excised inside-out patches, the current-voltage (I-V) relationships of the 9-pS channel were linear. In only 1 out of 47 active patches was the small-conductance Cl- channel still active 1 h after membrane excision. The addition of 0.1 microM of the catalytic subunit protein kinase A with 2 mM ATP to the cytoplasmic side restored channel activity in 8 out of 15 excised membrane patches. In 5 out of 467 patches of stimulated or nonstimulated cells, a larger Cl- conductance of 30 pS was also recorded. In excised inside-out patches this channel outwardly rectified and was activated by strong depolarization. In cultured DCTb cells, the small-conductance, cAMP-activated Cl- channel shares many properties with the cystic fibrosis transmembrane conductance regulator. Our results suggest that at least the small-conductance channel may participate in Cl- secretion across the apical membrane of DCTb in primary culture. This secretion may increase the rate of the apical Cl-/HCO3- exchange indirectly by enhancing the inwardly-directed Cl- gradient.

Effects of vasopressin on single Cl − channels in the apical membrane of distal nephron cells (A6)

We have investigated how two types of Cl − channels found in sodium transporting epithelium are regulated by arginine vasopressin (AVP). A6 cells cultured on permeable supports for 10 to 14 days have two types of Cl − channels in the apical membrane that have single channel conductances of 3 and 8 pS. In cells without AVP pretreatment, the 3 pS Cl − channel was more frequently observed than the 8 pS Cl − channel. AVP increased the open probability ( P o ) and single channel conductance of the 3 pS Cl − channel without significantly changing the P o or conductance of the 8 pS Cl − channel. On the other hand, AVP did not affect the number of the 3 pS Cl − channel, but increased the number of 8 pS Cl − channels. These observations suggest that AVP has two different pathways to increase apical membrane chloride conductance in distal nephron A6 cells; i.e., (1) increases the P o and single channel conductance of 3 pS Cl − channels and (2) increases the number of 8 pS Cl − channels.

Endothelin and vasopressin activate low conductance chloride channels in aortic smooth muscle cells.

The non-contractile aortic smooth muscle cell line A7r5 was used to study the membrane events involved in the effect of vasoconstrictor peptides. Whole-cell voltage-clamp and membrane potential recording techniques were used to demonstrate the contribution of an increased Cl- conductance to the late depolarization induced by endothelin-1 and vasopressin. During cell-attached patch recording with N-methyl-D-glucamine in the pipette, bath application of endothelin or vasopressin induced single-channel inward currents in the following minutes. The current/potential (I/V) curve of the most frequently observed channel type--a small conductance Cl- (SCl) channel--reversed near the cell membrane potential and showed a single-channel conductance of 1.8 pS for inward currents. After patch excision in an extracellular solution containing CaCl2 (2 mM), the frequency of SCl channel openings increased. Patch excision in the absence of peptide stimulation also produced this channel activity. Replacement of CaCl2 by a Ca2+ chelator on the intracellular face of a patch reversibly inhibited the channel activity, indicating that these SCl channels are Ca(2+)-activated Cl- channels. The single-channel I/V characteristic showed outward rectification above +50 mV. An analysis of the gating kinetics of the SCl channel is given. Another channel type was recorded less frequently after peptide stimulation. It had a lower conductance (1.0-1.3 pS) and slower kinetics and was designated a very small conductance Cl- channel. It is concluded that activation of two types of Cl- channels (at least one of which is Ca2+ dependent) is involved in the late depolarization produced by vasoconstrictor peptides in vascular smooth muscle cells of the aortic cell line A7r5.

Two types of chloride channel in the basolateral membrane of vestibular dark cells

Transepithelial and cell membrane potential measurements have suggested that the basolateral membrane of gerbil vestibular dark cells contains Cl − conductive pathways. We used the patch clamp technique to search this membrane for Cl − conductive channels which could account for the macroscopic observations. Two types of Cl − channel were found in both cell-attached and excised membrane patches. One type was found with an incidence of 19% and had a single-channel conductance of 95 ±1 pS ( N = 20) in symmetrical Cl − solutions. The other type was found with an incidence of 3% and had a large single-channel conductance of 360 ± 11 pS ( N= 12) in symmetrical Cl − solutions (LC-type Cl − channel). Both types of Cl − channel had linear current-voltage relations and at least 2 substates. In asymmetrical Cl − solutions (gluconate substitution) the current-voltage relations fit the Goldman-Hodgkin-Katz current equation for Cl −. Neither channel was blocked by Zn 2+, NPPB, DIDS, DNDS or quinine. The 95 pS channel exhibited a spontaneous ‘rundown’ of its activity within 1 to 10 min after being excised. This rundown was not reversed by the catalytic subunit of protein kinase A. Channel activity was not dependent on the presence of cytosolic Ca 2 + nor markedly altered by variations in cytosolic pH between 6.5 and 8.0. The two Cl − channels were distinguished by the membrane voltage ranges in which they were active and by their anion selectivity. The open probability of the 95 pS channel was insensitive to voltage and the anions NO − 3. 1 − and Br − were only half as permeable as Cl −. By contrast, the LC-type CI − channel was mostly active between about ±30 mV and equally permeable to NO − 3, I −, Br − and Cl −. The 95 pS Cl − channel may account for the observed transepithelial and intracellular voltage responses to Cl − concentration steps and provide the path for the recirculation of Cl − across the basolateral membrane. The LC-type Cl − channel shows the same lack of anion discrimination as the anion pathway activated during hyposmotic challenge.

A linear 16 pS Cl- channel in the apical membrane of Caco-2 cells.

A linear Cl- channel with a mean conductance of 16.3 +/- 0.4 pS (n = 7), as determined for inwardly directed currents in the cell-attached mode, was identified by the patch clamp technique in the apical membrane of Caco-2 cells in the early stage of postconfluential differentiation. This channel exhibited a low spontaneous activity (in 3 out of 137 experiments) and was not activated by strong polarization of the holding potential. After stimulation of the cAMP signal transduction cascade at different levels the 16 pS Cl- channel was activated in 7 out of 67 experiments during continuous cell-attached recording and remained active after excision of the patch. This channel differed markedly from both a linear low conductance Cl- channel and an intermediate conductance, outwardly rectifying (ICOR) Cl- channel, already reported for Caco-2 and other colonic cell lines. Thus the 16 pS channel may represent a further type of apical Cl- channel in colonocytes.

Microscopic elements of electrical excitation in Chara: Transient activity of Cl? channels in the plasma membrane

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl- as Ca2+ or Ba2+ (50 or 100 mM), but not as Mg2+ or K+ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl- channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl- channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl- channel activity was favored by voltages positive of the free running membrane voltage (> -45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activation of these channels. Neither type of Cl- channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca2+ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl- channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca2+ and Cl- for triggering and time course: Ca2+ uptake does not activate Cl- channels directly but first supplies a membrane-associated population of Ca2+ storage sites. Depolarization enhances discharge of Ca2+ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca2+ concentration ([Ca2+]cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca2+ buffer system. In turn, the transient rise in [Ca2+]cyt causes the transient activity of those Cl- channels, which are more likely to open at an elevated Ca2+ concentration.

Stretch-activated anion currents of rabbit cardiac myocytes.

1. Stretch-activated anion currents were studied in sino-atrial and atrial cells using the whole-cell patch clamp technique. With continuous application of positive pressure (5-15 cmH2O) through the patch clamp electrode, the cell was inflated and the membrane conductance was increased. 2. Voltage clamp steps revealed that the stretch-activated currents had time-independent characteristics. The increase in membrane conductance was reversible on subsequent application of negative pressure to the electrode. 3. The reversal potential of the stretch-activated currents was shifted by 60 mV for a 10-fold change in intracellular Cl- concentration, while it was unaffected by replacement of Na+ in the extracellular solution by N-methyl-D-glucamine. Cell superfusion with Cl(-)-deficient solution (10 mM Cl-) reduced the amplitude of outward current. These findings indicate that the stretch-activated conductance is Cl- selective. 4. The sequence of anion permeability through the stretch-activated conductance was determined to be I-(1.7) > NO3-(1.5) > Br-(1.2) > Cl-(1.0) > and F-(0.6). SCN- appeared to be more permeant than I-. 5. The stretch-activated conductance was reduced by the Cl- channel blockers, 4,4'-dinitrostilbene-2,2'-disulphonic acid disodium salt, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid or anthracene-9-carboxylate (9-AC). Administration of furosemide or bumetanide had no effect. 6. The stretch-activated Cl- current was recorded even though intracellular Ca2+ ions were chelated by including 10 mM EGTA in the pipette solution. Neither the specific peptide inhibitor of cyclic AMP-dependent protein kinase (50 microM), nor the non-selective blocker of protein kinases, H-7 (20 microM), was effective in reducing the stretch-activated Cl- current, suggesting that the stretch-activated Cl- current is a novel type of cardiac Cl- current, which shows a different modulatory mechanism from that of other cardiac Cl- currents.

Immunolocalization of chloride-transporting membrane vesicles in tracheal epithelial cells.

A membrane fraction eluted from a phenyl Sepharose column (MPS) was isolated from renal cortex and bovine tracheal epithelia that is enriched in a single type of Cl- channel [C. L. Preston, M. A. Calenzo, and W. P. Dubinsky. Am. J. Physiol. 263 (Cell Physiol. 32): C879-C887, 1992]. A 200-kDa membrane protein that copurifies with and appears to be specific to this fraction was purified and used to raise antisera for immunological characterization of these membranes. The antisera reacted in immunoblots with a 200-kDa protein in homogenates of bovine trachea, kidney, pancreas, lung, and intestine. There was also cross-reactivity with a 200-kDa protein in immunoblots in rat stomach, pancreas, and lung. There was no cross-reaction with rat skeletal muscle, cardiac muscle, or aorta. Thus this protein appears to be preferentially enriched in epithelial tissues. Examination of each major fraction during the purification of MPS membranes from trachea shows no enrichment of the 200-kDa protein in plasma, mitochondrial, or nuclear membrane fractions. The only significant enrichment was observed in MPS that is purified by hydrophobic chromatography. In frozen sections, antisera and monospecific immunoaffinity-purified antibodies localize the protein primarily to the apical domain of tracheal columnar epithelial cells with small punctate structures throughout the cytoplasmic compartment.

Chemiluminescence in the crude extracts of soybean seedlings. Postulated mechanism on the formation of hydroperoxide intermediates

It has been reported that weak chemiluminescence (CL) from crude extracts of soybean seedlings is remarkably enhanced with the addition of various aldehydes (Biochim. Biophys. Acta 1058, 209-216). The reactivity of certain emitter(s) with oxygen species was examined in the autoclaved extracts of seedling. When samples were reduced by the addition of hydrosulfite, two different types of reactivities in CL were defined. One type showed an initial rapid increase and a subsequent fast decay in CL upon mixing with oxygen. This rapid increase in CL intensity was independent of the presence of aldehydes, and was significantly suppressed by SOD. However, the subsequent slow decay phase in CL was dependent on the presence of aldehydes. In the sample reduced more moderately by borohydride, the same slow decay of CL appeared upon mixing with acetaldehyde and oxygen. This second type of Cl was not inhibited by active oxygen scavengers. Hydrogen peroxide added to unreduced (oxidized) samples also elicited CL. Three types of primary emitters may be oxidized to form transient hydroperoxide, and excited for light emission by slightly different ways: two of them are excited by abstraction of one atomic oxygen from the hydroperoxy intermediate with aldehyde or hydrogen peroxide, leading to formation of an excited hydroxide intermediate. The third is excited directly on the binding of superoxide anion to the reduced primary emitter.