Characterization of ion and fluid transport in human bronchioles

The regulation of the volume and composition of airway surface liquid is achieved through epithelial ion transport processes. In humans, these processes have been characterized in proximal but not distal airways. Segments of human bronchioles were dissected from surgically removed lung pieces. The transmural potential difference of microperfused bronchioles was inhibited by luminal exposure to amiloride and increased when exposed to the Cl secretagogues forskolin and ATP in the presence of amiloride. Human bronchiolar epithelial cells were cultured on permeable supports and studied in Ussing chambers. They generated a short circuit current (Isc) that decreased in response to amiloride and increased in response to forskolin and to ATP in the presence of amiloride. In low-Cl Kreb's Ringer bicarbonate, the baseline Isc and amiloride-induced decrease in Isc were not different, whereas the forskolin- and ATP-induced increases in Isc were smaller. Fluid transport measurement in excised bronchioles revealed a basal absorptive flow that was reduced by amiloride, whereas forskolin and ATP combined induced a secretory flow in the presence of amiloride. We conclude that human bronchioles actively absorb Na and fluid in unstimulated conditions and are capable of active Cl and fluid secretion when exposed to forskolin and to ATP.     

Mechanisms and receptor subtypes involved in the stimulatory action of endothelin-1 on rat adrenal zona glomerulosa

Endothelin (ET)-1 is the prototype of a family of 21-amino acid residue hypertensive peptides, acting through two subtypes of receptors, named ETA and ETB. ETs and their receptors are expressed in the adrenal cortex and medulla, and ET-1 enhances both corticosteroid and catecholamine release. ET-1 concentration-dependently (from 10(-11) to 10(-8) M) increased aldosterone secretion of both dispersed rat zona glomerulosa (ZG) cells and adrenal slices containing a core of medullary chromaffin tissue, but the response of the latter preparations was significantly more intense than that of the formers. The stimulatory effect of 10(-8) M ET-1 on dispersed ZG cells was blocked by the ETB-receptor antagonist BQ-788 (10(-7) M), but not by the ETA-receptor antagonist BQ-123 (10(-7) M); conversely, both ET-receptors antagonists counteracted aldosterone response of adrenal slices to ET-1. The -adrenoceptor antagonist l-alprenolol (10(-6) M) did not affect aldosterone response of dispersed ZG cells to ET-1 (10(-8) M), but it significantly lowered that of adrenal slices. l-Alprenolol also counteracted the aldosterone response of adrenal slices to the pure activation of ETB or ETA receptors, as obtained by using the selective ETB-receptor agonist BQ-3020 (10(-8) M) or ET-1 (10(-8) M) plus BQ-788 (10(-7) M). ET-1 concentration-dependently (from 10(-9) to 10(-8)/10(-7) M) stimulated catecholamine release by adrenal slices, and the effect was counteracted by both BQ-123 and BQ-788 (10(-7) M). Collectively, our findings suggest that, when the integrity of adrenal tissue is preserved, a two-fold mechanism underlies the aldosterone secretagogue action of ET-1 in the rat: i) a direct mechanism mediated by ETB receptors located on ZG cells; and ii) an indirect mechanism involving the ETA and ETB receptor-mediated local release of catecholamines, which in turn stimulate ZG cells in a paracrine manner.     

Effect of ion transport inhibitors and methacholine on short-circuit current of isolated guinea pig nasal epithelium

To clarify the ion/water secretion mechanism in the nasal epithelial cells, the Ussing chamber method was applied to the nasal mucosa isolated from guinea pigs. The preparation, which contained surface epithelial cells, showed a small but consistent potential difference between mucosal and submucosal sides (mucosal surface negative to submucosa). The short-circuit current (Isc) across the epithelial layer was measured, and the effects of Na+ and/or Cl- transport inhibitors and methacholine (MCh) on Isc were analyzed. The basal Isc was almost totally suppressed by the combined application of amiloride (Na+ transport inhibitor) and low-Cl- Krebs Ringer (KR) solution or solutions containing Cl- transport inhibitors (furosemide or DPC). The application of MCh elicited triphasic Isc responses, i.e., initial transient increase (phase 1) followed by a small decrease (phase 2) and further sustained increase (phase 3) in Isc. A possible ionic mechanism underlying phase 1 and 3 responses was analyzed. The Phase 1 response was greatly reduced by low-Cl- KR solution or furosemide but not influenced by amiloride. The Phase 3 response was augmented by amiloride and suppressed by low-Cl- KR solution, furosemide or DPC. These findings indicated that the basal Isc was associated with Cl- secretion and/or Na+ absorption across epithelial cells under short-circuit condition and that MCh increased Isc probably via enhancing Cl- secretion in the nasal surface epithelial cell.     

Signaling pathways involved in the A and B receptor-mediated cortisol secretagogue effect of endothelins in the human adrenal cortex

Endothelins (ETs) are a family of 21-amino acid hypertensive peptides, which together with their receptors ETA and ETB are expressed in human adrenal cortex. Evidence has been provided that ETs exert a potent secretagogue effect on human adrenocortical cells, acting through both ETA and ETB receptors. Therefore, it seemed worthwhile to study the signaling cascades mediating the cortisol secretagogue effect of the two receptor subtypes. Normal adrenal glands were obtained from consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Dispersed zona fasciculata-reticularis (ZF/R) cells were obtained by collagenase digestion and mechanical disaggregation. The selective activation of ETA and ETB receptors was obtained by exposing dispersed cells to ET-1 plus the ETB receptor antagonist BQ-788 and to the selective ETB receptor agonist BQ-3020, respectively. ETA and ETB receptors about equally contributed to the cortisol response of dispersed ZF/R cells to ETs. The phospholipase (PL) C inhibitor U-73122 abolished ETA-mediated secretory response, but only partially prevented the ETB-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes, while the Ca(2+)-channel blocker nifedipine was ineffective. The ETB receptor-, but not the ETA receptor-mediated cortisol response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. The inhibitors of adenylate cyclase, PKA, tyrosine kinase and lipoxygenase did not affect the secretory response to the activation of either receptor subtype. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZF/R cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate cortisol secretion from human ZF/R cells, acting through ETA receptors exclusively coupled with PLC/PKC-dependent pathway and ETB receptors coupled with both PLC/PKC- and COX-dependent cascades.     

Buffering action of endogenous nitric oxide on the adrenocortical secretagogue effect of endothelins in the rat

The secretagogue effect of endothelins (ETs) on the rat adrenal cortex is mediated by the ETB receptor. ETB receptors are coupled with nitric oxide (NO) synthase (NOS), and NO is known to inhibit steroid-hormone secretion from adrenal cortex. We investigated whether ETB-mediated NO production interferes with the stimulatory action of ETs on rat adrenal cortex. The selective agonist of ETB receptor BQ-3020 concentration-dependently increased aldosterone secretion from dispersed zona glomerulosa (ZG) cells and corticosterone secretion from dispersed zona fasciculata-reticularis (ZF/R) cells, and the NOS inhibitor NG-nitro-L-arginine methylester (L-NAME) potentiated the effect of BQ-3020 in a concentration-dependent manner. The guanylate cyclase inhibitor Ly-83583, at a concentration suppressing guanylin- and L-arginine-induced cyclic-GMP release from dispersed adrenocortical cells, did not affect the secretory response of ZG and ZF/R cells to BQ-3020. ET-1, an agonist of both ETA and ETB receptors, stimulated the release of both aldosterone and corticosterone by in situ perfused rat adrenal gland. This effect was potentiated by L-NAME and unaffected by Ly-83583. Collectively, our findings allow us to suggest that endogenous NO exerts in vivo and in vitro a cyclic-GMP-independent buffering action on the ETB receptor-mediated adrenocortical secretagogue action of ETs.     

Endothelin-3 at low concentrations attenuates inflammatory responses via the endothelin B2 receptor

Objective

The aim of this study was to clarify a role of endothelins (ETs: ET-1, -2, and -3) and their receptors (ETA, ETB1, and ETB2) in inflammatory responses.

Methods

Male Wistar rats (180–220 g) were used. The effects of ETs in the absence or presence of the ETA antagonist BQ-123/the selective ETB2 antagonist BQ-788, and the effect of the selective ETB1 agonist IRL-1620 and the nonselective ETB agonist BQ-3020, on rat hind paw oedema induced by several proinflammatory substances were examined. The involvement of nitric oxide (NO) in the effects of ETs on the paw oedema was investigated using the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME).

Results

ET-3, which acts mainly on ETB, at low concentrations specifically inhibited platelet-activating factor (PAF)-induced paw oedema, whereas neither ET-1 nor ET-2, both of which act on ETA and ETB, showed inhibitory activity. The inhibition by ET-1 and ET-3 (each 0.5 pmol/paw) in the presence of BQ-123 (66.4 ± 6.7 % and 65.4 ± 22.6 %, respectively), was comparable to that by ET-3 (0.5 pmol/paw) alone (65.4 ± 10.9 %), whereas neither ET-1 nor ET-3 in the presence of BQ-788 showed inhibitory activity. BQ-3020 (0.5 pmol/paw) inhibited the oedema by 50.9 ± 6.0 %, whereas IRL-1620 showed almost no activity. Additionally, L-NAME markedly attenuated the inhibitory effects of ET-3 on PAF-induced paw oedema. These results indicate that ETB2 may mediate NO production and attenuation of PAF-induced inflammatory responses. Moreover, ET-3 (0.5 pmol/paw) inhibited the oedema induced by ET-1 at higher dose and zymosan by 76.6 ± 11.0 and 85.4 ± 13.6 %, respectively, indicating that ET-3 at lower concentrations inhibits the paw oedema induced by various inflammatory substances.

Conclusions

ET-3 at low concentrations may attenuate inflammatory responses via ETB2 activation and NO production.     

Effects of Ca2+ on ileal transport and electrically induced secretion

[Ca2+] affects nerves and target cells in stimulus-secretion coupling. In flux-chamber studies of full-thickness rabbit ileum, we determined the effects of 1) ethylene glycol bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) 1.25 mM, 2) verapamil 0.1 mM and nifedipine 0.1 mM, and 3) trifluoperazine 0.1 mM on ion transport and its response to electrical field stimulation (EFS). EGTA increased JClm leads to s, JNam leads to s, Cl absorption and conductivity (G), and reduced Isc. In the absence of EGTA, EFS increased transmural PD and Isc and caused secretion of Na and Cl. EGTA prevented the responses to EFS, but the Isc responses to aminophylline and to glucose were normal. Verapamil reduced the response of Isc and Cl transport to EFS. Nifedipine reduced Isc but not the Isc response to EFS. Trifluoperazine reduced Isc and almost eliminated the Isc response to EFS. EFS did not increase the tissue concentration of cAMP. We conclude: 1) low extracellular [Ca2+] enhances net Cl absorption; 2) extracellular Ca2+ is required for the response of ion transport to EFS; 3) cAMP does not mediate Isc response to EFS; and 4) Isc response to EFS is blocked by trifluoperazine. The findings suggest that EFS stimulates secretion by increasing Ca entry into the epithelial cells, either directly or indirectly.     

Endothelin-1 inhibits mucin secretion from ovine airway epithelial goblet cells

Mucus hypersecretion is a feature of several respiratory diseases and frequently leads to obstruction of small airways where the principal source of mucous glycoproteins (mucins), the major macromolecular constituents of mucus, are goblet cells. Hence, inhibition of mucin secretion from these cells may be clinically beneficial. In this study, we have developed a lectin-based assay for mucin secretion from ovine airway goblet cells and used this assay to investigate the regulation of these cells by endothelin (ET)-1. ET-1 inhibited baseline mucin secretion (maximum inhibition: 60.3 +/- 4.2%, 50% inhibitory concentration: 0.8 +/- 0.17 nM). This response was abolished by the ET(A) antagonist, BQ-123 (1 muM), but not by the ET(B) antagonist, BQ-788 (1 muM). ET-1 (1 muM) did not affect mucin secretion stimulated by ATP (100 muM) but secretion in response to ATP (10 muM) was inhibited by 63.3 +/- 11.8%. This response could be eliminated by BQ-123, but not by BQ-788. Radioligand binding and immunohistochemistry indicated the expression of both ET(A)- and ET(B)-receptors on the epithelium. In summary, ET-1, acting via ET(A)-receptors, inhibits baseline and ATP-stimulated mucin secretion from ovine airway goblet cells. This represents the first report of a physiologic mechanism for inhibiting airway goblet cell mucin secretion; an understanding of this mechanism may provide opportunities for the treatment of obstructive airways disease.     

Prostaglandin D2 increases Cl secretion across canine tracheal epithelium through cyclo-oxygenase stimulation and cAMP production.

Prostaglandin (PG) D2 is one cyclo-oxygenase product of arachidonic acid metabolites that may play a role in the pathogenesis of asthma. To determine the effect of PGD2 on ion transport by airway epithelium and its mechanism of action, we measured bioelectric properties of canine cultured tracheal epithelium under short-circuit conditions in vitro. PGD2 (10(-7) M) increased short-circuit current (Isc) from 5.5 +/- 1.2 to 14.1 +/- 2.9 microA cm-2 (means +/- SE, P less than 0.01) when added to the mucosal solution, and to 22.2 +/- 3.8 microA cm-2 (P less than 0.001) when added to the submucosal solution, an effect that was accompanied by the corresponding increases in transepithelial potential difference and conductance. These effects were dose-dependent. The PGD2-induced increase in Isc was not altered by preincubation of cells with autonomic antagonists (phentolamine, propranolol, atropine), the lipoxygenase inhibitor AA-861, the protein kinase C inhibitor H-7, or the Na channel blocker amiloride, but it was inhibited by each of indomethacin, piroxicam, the Cl channel blocker diphenylamine-2-carboxylate, the Cl transport inhibitor furosemide, and Cl-free medium. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels were dose-dependently increased by PGD2. These results suggest that PGD2 may selectively stimulate airway epithelial Cl secretion via cyclo-oxygenase- and cAMP-dependent pathway.     

Differential regulations between adenosine triphosphate (ATP)- and uridine triphosphate-induced Cl(-) secretion in bovine tracheal epithelium. Direct stimulation of P1-like receptor by ATP

Adenosine 5'-triphosphate (ATP) stimulates airway epithelial Cl(-) secretion in a complicated manner. We examined the difference between ATP- and uridine 5'-triphosphate (UTP)-induced responses of short-circuit current (Isc) in bovine tracheal epithelium treated with amiloride. Each nucleotide caused an increase in Isc composed of the first and second peaks, where the second peak induced by ATP was higher compared with UTP. The ATP-induced second peak was inhibited by the protein kinase (PK) A inhibitor H89, saturation of P1 receptor with adenosine, and the P1 receptor antagonist 8-p-sulfophenyltheophylline, but not by the Ca(2+) chelator ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin, the adenosine breakdown enzyme adenosine deaminase, the ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate, or saturation of P2Y2 receptor with UTP. Thus, the response is associated with PKA-dependent pathway via P1-like receptor but not with Ca(2+)-dependent pathway via P2Y2 receptor, and ATP degradation products do not contribute to this response. Further, stimulation of cells with ATP increased PKA activity. In addition, pretreatment with glybenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator, reduced the second peak of Isc induced by ATP but was without effect on that induced by UTP. Therefore, ATP stimulates glybenclamide-sensitive Cl(-) secretion, and this action is partly mediated by PKA-dependent pathway via P1-like receptor.     

Epithelial ion transport of human nasal polyp and paranasal sinus mucosa

Nasal cavity and paranasal sinus have various functions. However, little information is available on ion transport in these upper airway epithelia. In the present study, we measured the anion secretion and the anion channel activity to characterize the ion transport in epithelial cells prepared from human paranasal sinus mucosa (PSM) and nasal polyp (NP). To estimate the anion secretion and the anion channel activity, we measured the short-circuit current (Isc) and the transepithelial conductance (Gt) sensitive to NPPB (a Cl(-) channel blocker). The NPPB-sensitive Isc in PSM was larger than that in NP, correlating to the NPPB-sensitive Gt (Cl(-) channel activity). Forskolin stably elevated the NPPB-sensitive Isc associated with an increase in the NPPB-sensitive Gt in PSM and NP. UTP transiently stimulated the Isc associated with an elevation of Gt in PSM and NP. The stimulatory action of UTP on Isc and Gt was diminished by application of NPPB but not benzamil in PSM and NP, suggesting that UTP induced the NPPB-sensitive Isc (Cl(-) secretion) and Gt (Cl(-) channel activity). These observations suggest that in human PSM and NP, cAMP stably stimulates anion secretion by activating the Cl(-) (anion) channels, and that UTP just transiently elevates anion secretion via activation of some Cl(-) (anion) channels.     

Endothelin synthesis and receptors in porcine kidney

As insufficient information on the endothelin (ET) system in the porcine kidney is available at present, we investigated renal ET-1 synthesis and ET receptors in this species. Because ET specifically affects renal and glomerular haemodynamics and distal tubular reabsorption, we studied ET-1 synthesis in isolated glomeruli and in inner medullary collecting duct (IMCD) cells and preproET-1 mRNA in renal cortex, isolated glomeruli and papillary tissue. In addition, we characterized density and properties of ET receptors in membranes from isolated glomeruli and papillary tissue. In contrast to isolated IMCD cells, which synthesized 120 +/- 11 fmol h(-1) mg-1 protein of ET-1, no such synthesis was found with isolated glomeruli in our assay system. Nevertheless, with RT-PCR preproET(-1) mRNA was clearly present in renal cortex and glomeruli as well as in papillary tissue. Glomerular membranes were found to have ET receptors with Bmax of 1.6 +/- 0.2 pmol mg-1 protein and Kd of 311 +/- 33 pmol L(-1). Using BQ-123 (10-5 M), a specific blocker of ETA receptors, we found that 58% of total receptors are ETA receptors. Thus, presumably 42% are ETB receptors (Bmax 0.7 +/- 0.1 pmol mg-1 protein; Kd 429 +/- 110 pmol L(-1)). Bosentan (10-5 M), an ETA- and ETB-receptor antagonist, blocked all ET receptors in glomerular membranes. Papillary membranes showed ET receptors with Bmax of 2.1 +/- 0.2 pmol mg-1 protein and Kd of 137 +/- 11 pmol L(-1). In the presence of BQ-123 (10-5 M) we found that all receptors are ETB receptors (Bmax 2.3 +/- 0.4 pmol mg-1 protein; Kd 162 +/- 25 pmol L(-1)). Bosentan (10-5 M) again blocked all ET receptors in papillary membranes, thus confirming our previous finding that IMCD cells possess high-affinity ETB receptors mediating the diuretic effects of ET. Thus, in the porcine kidney the ET system may act in an autocrine/paracrine manner at the glomerular as well as at the IMCD level.     

Effects of endothelin-1 on the membrane potential and slow waves in circular smooth muscle of rat gastric antrum.

Electrophysiological effects of endothelin-1 (ET-1) on circular smooth muscle of rat gastric antrum were investigated by using intracellular membrane potential recording techniques. ET-1 (10 nM) caused an initial hyperpolarization of the membrane which was followed by a sustained depolarization. ET-1 also increased the frequency but not the amplitude of slow waves. In the presence of the endothelin type A (ETA) receptor antagonist, BQ123 (1 microM), ET-1 (10 nM) depolarized the membrane and increased the frequency of slow waves, but without the initial hyperpolarization. The selective endothelin type B (ETB) receptor agonist, sarafotoxin S6c (10 nM), also depolarized the membrane and increased the frequency of slow waves. In the presence of the ETB receptor antagonist, BQ788 (1 microM), ET-1 (10 nM) hyperpolarized the membrane. However, in the presence of BQ788, ET-1 caused neither the depolarization nor the increase in the frequency of the slow waves. The ET-1-induced hyperpolarization was completely abolished by apamin (0.1 microM). In the presence of apamin, ET-1 depolarized the membrane and increased the frequency of slow waves. The ET-1-induced depolarization was significantly attenuated by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM). The increase of the frequency by ET-1 was observed both in the presence and absence of DIDS. These results suggest that, ET-1 hyperpolarizes the membrane by the activation of Ca2+-activated K+ channels via ETA receptors, and depolarizes the membrane by the activation of Ca2+-activated Cl- channels via ETB receptors. ET-1 also appears to increase the frequency of slow waves via ETB receptors, however this mechanism would seem to be independent of membrane depolarization.     

Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated Cl(-) secretion in human airway epithelia

Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)-dependent Cl(-) conductance. Activation of Cl(-) secretion in airways depends on simultaneous activation of luminal Cl(-) channels and basolateral K(+) channels. We determined the role of basolateral K(+) conductance in cAMP- dependent Cl(-) secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na(+) conductance and defective cAMP-mediated Cl(-) conductance. In non-CF tissues, Cl(-) secretion was significantly inhibited by the chromanol 293B (10 micromol/liter), a specific inhibitor of K(V)LQT1 K(+) channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba(2+) (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 micromol/liter) simultaneously activated Cl(-) and K(+) conductance. The K(+) conductance was reversibly inhibited by Ba(2+) and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K(V)LQT1. We conclude that the K(+) channel K(V)LQT1 is important in maintaining cAMP-dependent Cl(-) secretion in human airways. Activation of K(V)LQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl(-) channel activity or alternative Cl(-) channels could help to circumvent the secretory defect.     

Modification of Na and Cl transport in canine tracheal mucosa by prostaglandins

We tested the effect of prostaglandins PGF2 alpha and PGE1 on the transport of 36Cl and 22Na by canine tracheal epithelium. Sheets of epithelium were mounted in Ussing chambers and short-circuited. Addition of PGF2 alpha to the mucosal side resulted in an increase of net Cl secretion from 0.71 +/- 0.41 to 2.40 +/- 0.67 mu eq . cm-2 . h-1 without significant effect on net Na absorption. Prostaglandin E1 on the mucosal side increased net Cl secretion from 1.36 +/- 0.31 to 2.69 +/- 0.35 and decreased Na absorption from 0.87 +/- 0.16 to 0.49 +/- 0.09. Indomethacin significantly depressed net Cl secretion from 1.36 +/- 0.36 to 0.57 +/- 0.22. Subsequent addition of PGE1 augmented net Cl secretion to 3.88 +/- 0.75. PGE1 did not enhance [14C]mannitol fluxes across this epithelium. Cellular levels of cAMP increased in response to PGE1 from 130 +/- 12.7 to 642 +/- 33.4 pmol . mg prot-1 . 10 min-1, whereas PGF2 alpha had no effect. These data suggest that although effects of PGF2 alpha and PGE1 are similar as pertains to net Cl secretion, they differ in their effects on Na transport and their capacity to increase cAMP levels. Alterations in Cl and Na transport in response to PGE1 are likely to be mediated, at least in part, by the adenylate cyclase-cAMP system. Furthermore, endogenous prostaglandins may have an important regulatory role in ion transport by airways epithelium.     

Sodium channel but neither Na(+)-H+ nor Na-glucose symport inhibitors slow neonatal lung water clearance.

Normal clearance of alveolar liquid following birth requires active Na transport; however, the contribution of Na channels, Na-H antiports, and Na-glucose symports is unknown. We demonstrated that intraalveolar instillation of amiloride (n = 6) or the more specific Na channel blockers benzamil (n = 13) or phenamil (n = 12) before the first breath impaired lung water clearance relative to control newborns (n = 34). Benzamil and phenamil were more potent than amiloride (P less than 0.05). Neither the Na-H antiport inhibitor dimethyl amiloride (n = 7) nor the Na-glucose symport inhibitor phloridzin (n = 7) impaired lung water clearance. Ion substitution experiments with fetal rat type II alveolar epithelia demonstrated that more than 95% of their resting or terbutaline-stimulated short circuit current (Isc) depended upon Na bathing their apical membrane. Isc was decreased by amiloride (IC50 of amiloride-sensitive Isc = 0.3 x 10(-6) M) and benzamil (IC50 of benzamil-sensitive Isc = 0.3 x 10(-7) M) but was unaffected by dimethyl amiloride (10(-4) M). We conclude that in vivo postnatal clearance of fetal lung liquid can be impaired by Na channel blockers and is unaffected by blockers of Na-H antiports and Na-glucose symports. Na transport in fetal type II cells has high affinity for amiloride, and these cells likely contribute to normal neonatal lung liquid clearance.     

Mechanism of calcium ionophore stimulated Cl secretion from frog skin glands

The aim of the present study was to investigate the mechanism by which the calcium ionophore A23187 stimulates Cl and water secretion from exocrine glands in the frog skin. The Cl secretion was visualized as changes in short-circuit current (SCC) in skins where the Na absorption was blocked by amiloride applied to the apical membrane. Measurements of A23187 stimulated ion fluxes showed that the ionophore induced a net secretion of Cl, Na and K. The active Cl secretion was enhanced more than the Na and K secretion, resulting in a net secretion of negative ions which closely resembled the A23187-stimulated SCC. The effect of A23187 was abolished in skins pretreated with indomethacin, implying the involvement of prostaglandins in the response. Furthermore, the effect of A23187 was inhibited in the presence of quinacrine, indicating that the activation of the cyclooxygenase pathway is dependent on phospholipase A₂ activity. In addition, the A23187, but not the arachidonic acid stimulated Cl secretion was abolished in the presence of trifluoperazine, suggesting that the effect of the ionophore may be mediated via a Ca²⁺-calmodulin-dependent step located before the activation of the cyclooxygenase. The net water flow and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and time-course of the water secretion were similar to the changes observed for the Cl secretion. The A23187 stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophyllin, indicating that the effect of A23187 was caused by an increase in the intracellular cAMP level in the gland cells. From the present data it is suggested that the calcium ionophore stimulates the Cl and water secretion from frog skin gland not by a direct effect of Ca²⁺-ions per se, but in an indirect manner by stimulating the prostaglandin synthesis, which probably results in an increase in the cAMP level in the gland cells.     

Ion transport by amphibian antrum in vitro. I. General characteristics.

Both Necturus and bullfrog antrum show stable PD, resistance, and short-circuit current (Isc) when mounted in an Ussing chamber. Measurements of Na+ and Cl minus flux showed that both ions are actively transported across Necturus antrum, Na+ from secretory to nutrient, Cl minus from nutrient to secretory (both net fluxes being similar to 0.30 mueq cm minus 2 h minus 1). Only the Na+ transport contributed to the Isc and PD as evidenced by a) Na+ removal, b) the effects of amiloride on the secretory surface, c) the effects of ouabain on the nutrient side. Microelectrode experiments confirm the Na+ conductance of the secretory cell membrance, a HCO3 minus conductance of both cell membranes, and a KCl conductance across the nutrient cell membrane. In addition, antrum apparently secretes alkali (similar to 0.35 mueq cm minus 2 h minus 1), which secretion is sensitive to metabolic inhibitors and Diamox. Nutrientside HCO3 minus increased the rate of alkaline secretion and a transmucosal HCO3 minus gradient could contribute to ISC and PD. A model is proposed to account for the electrical properties of the tissue.     

A novel bioactive 31-amino acid ET-1 peptide stimulates eosinophil recruitment and increases the levels of eotaxin and IL-5

OBJECTIVE AND DESIGN: Investigation of the role of a novel inflammatory mediator 31-amino acid endothelin-1 [ET-1 (1-31)], a major ET derivative in granulocytes, in eosinophil recruitment after its subcutaneous administration to mice. METHODS: Various ET-1 derivatives (100 pmol), with or without ET receptor antagonists (200 pmol), were administered subcutaneously to mice, and then the eosinophil migration into and chemokine levels in the injected loci were analyzed. RESULTS: ET-1 (1-31) and a 21-amino acid endothelin-1 (ET-1), but not big ET-1, induced eosinophil migration into the injected loci with a peak after administration for 12 h, and increased the levels of eotaxin and interleukin-5 with peaks at 6 and 24 h, respectively. These effects of ET-1(1-31) and ET-1 were significantly inhibited by an ETA receptor antagonist, BQ-123, but not by an ETB receptor antagonist, BQ-788. CONCLUSION: Novel bioactive ET-1 (1-31) induces local eosinophil migration, and increases in eotaxin and interleukin-5 through an ETA or ETA-like receptor.