Lysyl-bradykinin Research Articles

Bradykinin‐stimulated cyclooxygenase activity results in vas deferens anion secretion in vitro

Epithelia lining the vas deferens are thought to express high levels of cyclooxygenases (COX) 1 and 2 in the presence of testosterone, which may ultimately affect bicarbonate secretion into the duct lumen. When grown in culture, porcine vas deferens epithelial cells respond to lysylbradykinin (LBK) with an increase in short circuit current (Isc; indicating net anion secretion) and such effect is reduced by 85% when these epithelia are exposed to indomethacin, a non-selective COX inhibitor. Gene expression analysis reveals that both COX-2 and COX-1 are expressed in the cultured porcine vas deferens epithelia. Subsequent experiments revealed that PGE2, 1-OH prostaglandin E1 (EP4 receptor agonist) and butaprost (EP2 receptor agonist) concetration-dependently increase Isc, whereas sulprostone (EP1 and EP3 receptor agonist) produces no change in Isc. These results demonstrate that peptide hormones acutely modulate the luminal environment to which sperm is exposed in the vas deferens by acutely enhancing the production of prostaglandins. [NIH support RR-17686]

International Union of Pharmacology. XLV. Classification of the Kinin Receptor Family: from Molecular Mechanisms to Pathophysiological Consequences

Kinins are proinflammatory peptides that mediate numerous vascular and pain responses to tissue injury. Two pharmacologically distinct kinin receptor subtypes have been identified and characterized for these peptides, which are named B1 and B2 and belong to the rhodopsin family of G protein-coupled receptors. The B2 receptor mediates the action of bradykinin (BK) and lysyl-bradykinin (Lys-BK), the first set of bioactive kinins formed in response to injury from kininogen precursors through the actions of plasma and tissue kallikreins, whereas the B(1) receptor mediates the action of des-Arg9-BK and Lys-des-Arg9-BK, the second set of bioactive kinins formed through the actions of carboxypeptidases on BK and Lys-BK, respectively. The B2 receptor is ubiquitous and constitutively expressed, whereas the B1 receptor is expressed at a very low level in healthy tissues but induced following injury by various proinflammatory cytokines such as interleukin-1beta. Both receptors act through G alpha(q) to stimulate phospholipase C beta followed by phosphoinositide hydrolysis and intracellular free Ca2+ mobilization and through G alpha(i) to inhibit adenylate cyclase and stimulate the mitogen-activated protein kinase pathways. The use of mice lacking each receptor gene and various specific peptidic and nonpeptidic antagonists have implicated both B1 and B2 receptors as potential therapeutic targets in several pathophysiological events related to inflammation such as pain, sepsis, allergic asthma, rhinitis, and edema, as well as diabetes and cancer. This review is a comprehensive presentation of our current understanding of these receptors in terms of molecular and cell biology, physiology, pharmacology, and involvement in human disease and drug development.

COX-dependent and -independent pathways in bradykinin-induced anion secretion in rat epididymis.

Lysylbradykinin (LBK) added to the apical or basolateral side of cultured rat epididymal monolayers stimulated a rise in short-circuit current (Isc) due to anion secretion. The concentration-response relationships for the apical and basolateral applications have EC50 value of 0.001 microM. The responses to apical or basolateral application of LBK were blocked by WIN64338, a specific B2 receptor antagonist, but not by Des-Arg9,[Leu8]-BK, a specific B1 receptor antagonist, indicating that the LBK effects were mediated through B2 bradykinin receptors. Experiments to desensitize the B2 receptors by repeated stimulation have demonstrated that the responses to apical or basolateral LBK were due to discrete receptors on the apical or basolateral surface. In epithelia clamped in the Ussing chambers, addition of LBK to the apical or basolateral surface evoked release of PGE2 into the apical and basolateral bathing solutions over the first 10 min following hormone addition. LBK added to the basolateral side elicited a greater release than it was added to the apical side. Pretreatment of the epithelia with piroxicam (5 microM) abolished PGE2 release elicited by apical or basolateral LBK and abrogated the Isc induced by basolateral LBK. However, the rise in Isc induced by apical LBK was reduced by 31.3% only. The anion secretion response to apical LBK was not affected by MDL-12330A, an adenylate cyclase inhibitor, but greatly attenuated by thapsigargin, an inhibitor of intracellular Ca2+ release. However, the reverse effects were seen for basolateral LBK. It is concluded that distinct pathways are involved in the stimulation of anion secretion by apical or basolateral LBK. The response to basolateral LBK was COX-dependent, mediated by PGE2 and involves cAMP as second messenger. In contrast, the response to apical LBK is largely COX-independent, not mediated by PCE2 and involves Ca2+ as intracellular messenger.

Anion secretory effects of a non-peptide mimic of bradykinin (FR190997) on mouse colon epithelium

FR190997, a new non-peptide mimic was investigated using the chloride secretory response of the mouse colon as a test system. The increase in short circuit current (SCC) to FR190997 was approximately equal to that of lysyl bradykinin (LBK). It is shown that the current increase to FR190997 is due to electrogenic chloride secretion through an action at B2-kinin receptors and involves prostaglandin formation. In these respects its actions are identical to those of LBK, except that the responses to FR190997 are prolonged. However FR190997 produces a long lasting desensitisation both to itself and to LBK and it did not prove possible to protect against this using the high affinity antagonist at B2 receptors, Hoe 140. It is suggested that FR190997 either slows receptor recycling or leads to degradation of receptors, such that the reappearance of sensitivity may depend on new receptor synthesis.

Cleavage specificity of a myofibril-bound serine proteinase from carp ( Cyprinus carpio) muscle

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159–66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl–lysyl–bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.

Removal and restoration of epithelial chloride secretory activity of kinins by gene manipulation

Kinins are known to stimulate electrogenic chloride secretion in many mammalian epithelia, including those of the airways and the alimentary tract. In this study the chloride secretory activity of lysylbradykinin (LBK) on murine colonic epithelium has been examined, specifically to discover the primary and final effector mechanisms in this process, i.e. which kinin receptors are involved and which chloride channels are responsible for chloride secretion. The approach used was to modify the mice genetically and assess the effects on kinin mediated chloride secretion using voltage clamping at zero potential. Briefly, LBK increased SCC in mouse colon by ∼ 150 μA cm−2 with an EC50 of ∼ 5nM. In null CF mice LBK, 1μM had no effect on chloride secretion, but reduced SCC due to K+ secretion. This effect is normally masked in wild-type tissues by dominant chloride secretion, but can be shown to occur to the same extent by measuring K+ secretion with radioisotopes. Null CF mice produce no cftr, but CFTR was introduced into CF mice by injecting a YAC containing the human CF gene into the pronucleus of CF zygotes. Colonic epithelia from mice with the incorporated YAC showed the same sensitivity to LBK as wild-type tissues and achieved the same maximal chloride secretory response. Colonic epithelia from mice in which the B2r gene had been disrupted showed no response to LBK at normally supramaximally effective concentrations, although responses to other secretagogues were normal. Similarly des-Arg-BK caused no acute chloride secretory response in colonic epithelia from B2 knockout mice, however small responses appeared if tissues were incubated in vitro for 3–6 h. It is concluded that cftr chloride channels and B2rs are required for electrogenic chloride secretion. Further CFTR can replace cftr with no effect on either the sensitivity or extent of chloride secretion. In vitro, colonic epithelia may generate B2rs which, upon activation, have a minor effect on chloride secretory activity.

Degradation of lysylbradykinin by endopeptidase 24.11 and endopeptidase 24.15

Lysylbradykinin (LBK), a potent bioactive peptide with pleiotropic actions, is the major kinin generated in the extravascular space. To explore possible mechanisms of inactivation of this peptide in tissues, we evaluated its degradation by endopeptidase 24.11 (EP 24.11) and endopeptidase 24.15 (EP 24.15), two zinc metalloenzymes widely distributed in tissues. EP 24.11 cleaved LBK at the Gly5-Phe6 and Pro8-Phe9 bonds, whereas EP 24.15 cleaved the Phe6-Ser7 bond. Determination of kinetic constants for degradation of LBK by the two enzymes yielded kcat/Km ratios of 5.2 x 10(5) and 8.4 x 10(5) for EP 24.15 and EP 24.11, respectively, indicating that LBK is a good substrate for both enzymes. The findings demonstrate that both EP 24.11 and EP 24.15 efficiently degrade LBK and thus may contribute to the inactivation of this peptide in tissues.

Cyclic AMP and Ca2+ interactions affecting epithelial chloride secretion in human cultured colonic epithelia

1. Chloride secretion in three types of cultured epithelial monolayers derived from a single human colonic adenocarcinoma was measured in terms of short circuit current. The three cell types were designated HCA-7, Colony 3 and Colony 29. 2. Responses of HCA-7 monolayers to basolaterally applied lysylbradykinin (LBK) (10-1000 nM) or carbachol (1-100 microM) were potentiated by pre-exposure to forskolin (10 microM) for 5 min. Forskolin itself increased short circuit current (SCC), so that the total response to forskolin and LBK or carbachol were non-additive. 3. Colony 3 cells did not respond to LBK on either face but did to carbachol on the basolateral side, while Colony 29 epithelia responded to LBK on both sides and to carbachol and histamine basolaterally. Unlike HCA-7 epithelia, responses in Colony 3 and Colony 29 epithelia were not potentiated by forskolin, but were attenuated by piroxicam. 4. In the presence of piroxicam, both forskolin and prostaglandin E2 were able to potentiate the action of both LBK and carbachol in Colony 29 epithelia. 5. LBK receptor activation in Colony 29 epithelia is transduced into an increase in intracellular Ca2+ and cyclic AMP, while in HCA-7 epithelia there is only an increase in intracellular Ca2+ (Cai). These conclusions are considered to apply to both apical and basolateral kinin receptors. 6. It is shown that forskolin has no effect on the elevation of Ca2+ by LBK in either HCA-7 or Colony 29 cells. 7. It is concluded that potentiation of agonist responses occurs when cyclic AMP is raised at the time that intracellular Ca2+ increases. No potentiation of LBK or carbachol by forskolin occurs in Colony 29 monolayers as these agonists increase cyclic AMP via eicosanoid production.

Kinin-induced chloride permeability changes in colony 29 epithelia estimated from 125I- efflux and MEQ fluorescence.

1. The changes in apical Cl- permeability of Colony 29 human colonic epithelial monolayers were estimated from the rate constant of 125I- efflux from tissues loaded with the isotope. 2. Forskolin was used to increase intracellular concentrations of adenosine 3:5' cyclic-monophosphate (cyclic AMP), and A23187 to increase intracellular free Ca2+ (Cai). Both treatments increased the rate constant for 125I- efflux, indicating an increase in apical Cl- permeability. 3. Lysylbradykinin (LBK) also increased the rate constant for 125I- efflux, sometimes biphasically. Chelation of intracellular Ca2+ with BAPTA or prevention of prostaglandin formation with piroxicam, attenuated but did not eliminate the effect of LBK. It is concluded that LBK affects 125I- efflux through the agency of both cyclic AMP and Ca2+. 4. Ba2+ attenuated the effect of LBK and A23187 on 125I- efflux, but had no effect on the action of forskolin. It is concluded that Ca2+ has a major effect on K+ channels, the resulting hyperpolarization increasing the driving force for 125I- efflux. A secondary effect on Ca(2+)-sensitive Cl- channels is possible. By contrast, cyclic AMP exerts it major effect on apical Cl- channels. 5. Using a Cl- sensitive fluorescent dye, MEQ, the intracellular chloride concentration, Cli was estimated to be around 30 mM, which was increased to around 50 mM by forskolin, suggesting cyclic AMP could activate the Na-K-2Cl co-transporter. 6. MEQ fluorescence was used to estimate Cl- influx and efflux rates of epithelial cells. These were increased three fold by forskolin and dibutyryl cyclic AMP and two fold by LBK and histamine. 7. It is concluded that LBK increases electrogenic chloride secretion in Colony 29 epithelia through the generation of second messengers cyclic AMP and Ca2+, each of which may act on both apical and basolateral membranes.

Activation of ion channels by lysylbradykinin in the HCA‐7 colony 29 human adenocarcinoma cell line

1. The patch-clamp technique, both cell attached and inside-out patches, was used to examine the effects of lysylbradykinin (LBK) and A23187 on ion channels in cultured Colony 29 epithelial cells derived from a human adenocarcinoma. 2. LBK and A23187 applied directly to the intact cell stimulated the opening of a number of types of ion channel including Ca(2+)-activated K+ channels. 3. By use of inside-out patches, anion channels could be stimulated to open by application of protein kinase A and ATP to the cytosolic surface. Ca(2+)-activated K+ channels were also identified in isolated membrane patches. 4. The results suggest that the anion secretion which is stimulated by LBK is a complex event, involving the activation of a number of different types of ion channel, and that part of the response is the result of hyperpolarization of the cell by activation of Ca(2+)-activated K+ channels. From the data presented in this and the accompanying papers it appears that the Ca(2+)-sensitive K+ channels would be equally effective in either the apical or basolateral membranes.

Differential antagonistic effect of hydroalcoholic extract from Hymenaea martiana hayne arzeik on kinin and other agonist‐induced contractions of the isolated rat uterus and Guinea‐pig ileum

AbstractThis study was undertaken to evaluate the action of the hydroalcoholic extract (HE) from the bark of Hymenaea martiana on bradykinin (BK), lysyl‐bradykinin (L‐BK), acetylcholine (ACh), angiotensin II (AII), prostaglandin F2a (PGF2a), serotonin (5‐HT), oxytocin (Ot) and histamine (His)‐induced contractions of the isolated rat uterine muscle and guinea‐pig ileum. The HE (50–200 μg/mL) added to the bath for 20 min caused a concentration‐dependent rightward displacement of BK, L‐BK and ACh‐induced contractions in the rat uterus, allied to a discrete but significant reduction of maximal responses to the latter two agonists. By contrast, at the same range of concentrations the HE antagonized in a concentration‐dependent but noncompetitive manner the contractions induced by AII, but only at high concentrations (200 μg/mL) it significantly inhibited contractions evoked by both PGF2a and Ot, while contractile responses induced by 5‐HT were not affected. In the guinea‐pig ileum, the HE of H. martiana (50 and 100 μg/ml) caused a discrete rightward displacement of the BK and ACh concentration—response curves. Higher concentrations of the HE of H. martiana (200 μg/mL) caused a marked depression of BK and ACh‐induced maximal responses. These findings show that the active principle(s) presents in the HE from the bark of H. martiana exhibits an interesting pharmacological profile against several neurotransmitter‐induced contractions in nonvascular smooth muscles. Such actions may be relevant for supporting, at least in part, the use of this plant in folk medicine.

Chloride channels and anion fluxes in a human colonic epithelium (HCA-7).

1. Colonic epithelial cells, derived from a human adenocarcinoma (HCA-7), were examined by the patch clamp technique. 2. Outwardly rectifying anion (Cl-) channels were identified in the apical membrane. The conductance was g(in) approximately 26 pS, g(out) approximately 40 pS. The open state probability of the channels increased with depolarization and the selectivity for Cl- over K+ (PCl/PK) was approximately 7.5. 3. The channels were sensitive to intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP, 0.1 mM), but not to Ca2+ (at concentrations up to 1 mM). At depolarized potentials the channels were blocked by pirentanide (1-5 microM) applied intracellularly. 4. HCA-7 monolayers loaded with 125I- (as a marker for Cl-) were used to measure I- efflux and converted to instantaneous rate constants. 5. The rate constant for I- efflux was increased by forskolin and lysylbradykinin (LBK). The effects of forskolin were not effected by BAPTA (an intracellular calcium chelator). The effects of LBK were inhibited by BAPTA and by Ba2+, indicating that LBK raised intracellular Ca2+ (Cai) which activates Ca(2+)-sensitive K-channels, the latter being blocked by Ba2+. 6. Although it cannot be conclusively proved that the outwardly rectifying chloride channels described here are solely or partially responsible for the increased anion efflux or transepithelial chloride secretion, the channels are likely to be more relevant for cyclic AMP-requiring rather than Ca(2+)-requiring secretagogues.

Relation of anion secretory activity to intracellular Ca 2+ in response to lysylbradykinin and histamine in a cultured human colonic epithelium

A cultured human epithelial cell line, Colony 29, has been used to investigate the relation between anion secretion and intracellular Ca 2+ concentration (Ca i) in response to the secretagogues, lysylbradykinin (LBk) and histamine. Anion secretion was measured as short-circuit current (SCC) responses in epithelia cultured on pervious supports. Ca i was measured both in cell suspensions and epithelial monolayers using Fura-2 fluorescence. While it is concluded that raised Ca i is responsible for anion secretion the relationship is complex. For both secretagogues there is a receptor reserve, that is the maximal Ca i increase is greater than that required to cause a maximal secretory response. By examining the interactions between maximally effective concentrations of LBk and histamine it was shown that neither the SCC nor Ca i responses behaved additively. From observations in the absence of external Ca 2+ it was concluded that both secretagogues cause Ca 2+ release from the same intracellular source, but that in normal conditions Ca 2+ derived from intracellular and extracellular sources is responsible for the full effect.

Separate agonist‐specific oscillatory mechanisms in cultured human sweat duct cells.

1. Cultured sweat duct cells (CSDCs) were grown to confluency on a permeable support, and the pharmacological ion transport regulation was assayed by transepithelial voltage clamp techniques. 2. Exposure of the serosal membrane of CSDCs to methacholine (MCh), lysylbradykinin (LBK) or histamine produced an oscillating short-circuit current (Iscc) response, which could be divided in an initial transient phase and a sustained oscillating phase, the latter of which was totally dependent on external Ca2+. 3. The Iscc responses evoked by LBK and histamine were, in contrast to the cholinergic response, characterized by a marked desensitization and short duration of the subsequent phase of Iscc oscillations. 4. Prolonged Iscc oscillations, reflecting continuous Ca2+ influx, were seen following MCh stimulation, and in response to LBK or histamine stimulation, when cells had been pre-treated with MCh. This pre-treatment effect of MCh was independent of continuous muscarinic receptor occupation, and it was unrelated to nicotinic receptor occupation. 5. It is suggested that MCh stimulation selectively initiates an influx of Ca2+ to an intracellular pool, from where Ca2+ can be discharged repetitively. In contrast, LBK and histamine only activate discharge of Ca2+ from such an intracellular pool, resulting in a limited response, given no prior stimulation by MCh of the Ca2+ influx mechanism.

Action of pregnane compounds from Mandevilla illustris against contractions induced by kinins and other oxytocics in the rat isolated uterus

1. The effects of 5 pregnane compounds isolated from the rhizomes of Mandevilla illustris were examined against bradykinin (BK), Lysyl-bradykinin (L-BK), acetylcholine (ACh) and oxytocin (Ot)-induced contractions in the isolated uteri of the rat. 2. Compounds MI 15 and MI 18 (5–40 μg/ml) caused a parallel and concentration-dependent rightward displacement of BK and L-BK concentration-response curves. Compound MI 21 (2.5–10 μg/ml) also produced a concentration-dependent displacement to the right of the BK concentration-response curve, but reduced its maximal response. Schild analysis of these data were linear ( r close to l) and furnished the following PA 2 values (as G/ml): 6.0, 5.1 and 5.9, respectively. However, the slopes were significantly higher than unity. Compounds MI 25 and MI 27 (10–40 μg/ml) caused little or even no effect against BK and ACh responses. 3. In addition, compounds MI 18 and MI 21 (10–40 μg/ml) also antagonized in a concentration-dependent manner L-BK concentration-response curves. Schild plot were linear ( r close to l) and yielded the nominal pA 2 values (as G/ml) of 5.0 and 5.8, respectively, but the slopes were significantly different from one. 4. Like the results obtained previously with the crude extract from M. illustris, the purified compounds from the rhizome of this plant were not selective towards kinin action since at the same range concentrations they markedly interfered with both the sensitivities and the maximal responses caused by ACh and Ot in this preparation.

Antagonism of kinin-induced contraction of isolated rat uterus by the crude hydroalcoholic extract from Mandevilla illustris

1. 1. The crude aqueous/alcoholic extract (CE) of Mandevilla illustris (Apocynaceae) rhizomes was analysed against contractile response elicited by bradykinin (BK), lysyl-bradykinin (L-BK), oxytocin(Ot), acetylcholine (Ach), angiotensin II (AII) and barium chloride (BaCl 2) in the isolated uterus of the rat. 2. 2. The CE of this plant (0.5–2.0 mg/ml) caused a parallel and concentration-related rightward displacement of BK and L-BK contractile responses. Schild plot revealed a linear relationship ( r close to one) and yielded nominal PA 2 values of 3.6 and 3.2 respectively, but the slopes were significantly different from unity. 3. 3. However, the anti-BK action of the CE of M. illustris was not selective to kinin action, since in the same range concentration the CE also affected uterine contractile responses induced by Ot, Ach, AII and BaCl 2.

Differential effects of lysyl bradykinin and thapsigargin on epithelial chloride secretion

Differential effects of lysyl bradykinin and thapsigargin on epithelial chloride secretion

Inhibitory Effects of Kinins on Angiotensin I Conversion in the Local Circulation

The inhibition of angiotensin converting enzyme (ACE) by kinins was studied using bradykinin (BK), lysyl-bradykinin (Lys-BK), [Hydroxyproline3]-bradykinin ([Hyp3]-BK) and [Hydroxyproline3]-lysyl-bradykinin ([Hyp3]-Lys-BK). The latter two are novel kinins recently identified in our laboratory. All the four kinins displayed competitive inhibition on the conversion of angiotensin I to angiotensin II by purified canine lung ACE. Inhibition constants (Ki) for the four kinins were estimated from Dixon's plot as follows-BK: 0.27 microM, Lys-BK: 0.57 microM, [Hyp3]-BK: 0.34 microM, and [Hyp3]-Lys-BK: 0.27 microM. In the rat hindlimb perfusion system, the kinins were demonstrated to partially inhibit angiotensin I conversion to angiotensin II by the vascular ACE. Taken together, these results suggest that angiotensin II formation by ACE in the vascular tissue is possibly inhibited by local kinins, especially after ACE inhibitor administration. This indirect action of kinins, coupled with its direct vasodilatory action, might indicate a cooperative participation in vasodilation.

The kallikrein-kinin system in inflammation.

The kallikrein kinin system is shown diagrammatically in Figure 1 and indicates the various pathways by which kinin (bradykinin or lysyl bradykinin) can be formed. The intrinsic coagulation-kinin system is depicted on the left, and the tissue kallikrein-kinin pathway on the right. An up-to-date exposition of the various reactions of the intrinsic-coagulation pathway has been published in a recent review. In brief, it is initiated by Hageman factor autoactivation 2,3 conversion of prekalli-krein to kallikrein 4,5 a positive feedback in which Hageman factor is rapidly cleaved8by kallikrein 6,7 and cleavage of high molecular weight (HMW)-kininogen8. The prekallikrein is complexed to HMW-kininogen in plasma via the HMW kininogen light chain 9,10 The tissue pathway, by contrast, is dependent upon local secretion of tissue kallikrein by cells followed by digestion of low molecular weight (LMW)-kininogen to release lysyl-bradykinin; the latter peptide is rapidly converted to bradykinin by a plasma aminopeptidase 11 The source of kininogen available to the tissue system is dependent either upon local accumulation of plasma or kininogen that is present in interstitial fluid.KeywordsAllergy Clin ImmunolKinin ActivityHereditary AngioedemaTissue KallikreinPlasma KallikreinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Human eccrine sweat gland epithelial cultures express ductal characteristics.

1. Isolated human eccrine sweat glands were cultured in vitro. Cells were harvested and plated onto permeable supports to form confluent cell sheets, area 0.2 cm2. These were used to study the electrogenic transepithelial transport of ions by measurement of short-circuit current (SCC). Epithelial sheets had a basal SCC of 5.89 +/- 0.62 microA cm-2 (n = 33) and a transepithelial resistance of 74.1 +/- 5.6 omega cm2 (n = 33). The transepithelial potential difference varied between -0.2 and -1.8 mV with a mean value of -0.71 +/- 0.09 mV (n = 33). 2. The basal current was abolished by addition of 10 microM-amiloride to the apical bathing solution. The concentration of amiloride which inhibited basal SCC by 50% (EC50) was 0.4 microM. Cultures prepared from the secretory coil of sweat glands, rather than from whole glands, were similarly sensitive to amiloride (EC50 = 0.8 microM). 3. Lysylbradykinin (LBK), carbachol, isoprenaline, prostaglandin E2 (PGE2) and A23187 all increased SCC in cultures from whole glands. LBK responses were obtained with basolateral and not with apical application. Furthermore LBK actions were not substantially altered by cyclo-oxygenase inhibition but showed marked desensitization upon repeated application. Sheet cultures prepared from sweat gland coils also showed SCC responses to both carbachol and LBK. Forskolin, an activator of adenylate cyclase, did not alter SCC in either type of preparation. 4. Replacement of chloride and of chloride and bicarbonate in the bathing solution did not cause attenuation of the responses to LBK or carbachol in whole-gland sheet cultures. Furthermore responses were unaffected by piretanide or acetazolamide. These results were taken to indicate that anion secretion was not the basis for the SCC responses. 5. Responses to LBK and carbachol were significantly reduced by amiloride (10 microM), this effect being reversible. No responses to LBK or carbachol were seen when N-methyl-D-glucamine (NMDG) was used to replace sodium, whereas reintroduction of sodium ions restored responsiveness to these agents. 6. The SCC responses to the muscarinic agonist carbachol and to LBK appear to be due to stimulation of amiloride-sensitive, electrogenic sodium absorption in whole-gland sheet cultures. Further it would appear that, in culture, the pleuripotential capacity of the cells is revealed since both whole-gland and secretory coil cultures exhibit some properties usually associated in vivo with duct cells. Many mammalian epithelia show electrogenic chloride secretion both in response to carbachol and LBK but also in response to activation of adenylate cyclase with forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)