kallidin and Cystic-Fibrosis

The short-circuit current technique was used to examine the effects of N2-L-lysylbradykinin (LBK) on chloride secretion in the mucosae of the mouse intestine. It was found to be a potent chloride secretagogue in the mucosa lining the colon, jejunum, and cecum, as it is in most mammals, with 2 nM being sufficient to cause half-maximal secretion. The extent of the responses was in the order cecum > colon > jejunum. In cystic fibrosis (CF) null mice, with no CF transmembrane conductance regulator (CFTR) chloride channels, LBK caused no chloride secretion, but transporting activities for other ions were revealed. Introduction of the human CF gene into the genome of CF null mice at the zygote stage restored the chloride secretory activity of LBK, with only minor differences in potency. In mice in which the kinin B2 receptor gene had been disrupted, LBK had no effect, whereas the responses to forskolin were unchanged. Thus the acute effects of kinins on chloride secretion depend uniquely on kinin B2 receptors and CFTR chloride channels, which form the primary and final effector mechanisms of the secretory process.

Topics: Animals; Chloride Channels; Chlorides; Chromosomes, Artificial, Yeast; Colforsin; Cystic Fibrosis; Humans; Intestinal Mucosa; Kallidin; Mice; Mice, Knockout; Receptor, Bradykinin B2; Receptors, Bradykinin

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 approximately 150 microA cm-2 with an EC50 of approximately 5 nM. In null CF mice LBK, 1 microM 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 B1rs which, upon activation, have a minor effect on chloride secretory activity.

Topics: Animals; Bradykinin; Chlorides; Chromosomes, Artificial, Yeast; Colon; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelium; Humans; In Vitro Techniques; Ion Transport; Kallidin; Kinins; Mice; Mice, Transgenic; Receptor, Bradykinin B1; Receptors, Bradykinin

1. The delta F508 mutation of the cystic fibrosis (CF) gene is of high frequency in man (1 in 25) and in homozygotes causes cystic fibrosis. It is suggested that cystic fibrosis heterozygotes withstand secretory diarrhoea better than normal individuals and so are genetically advantaged. This hypothesis has been examined by measuring electrogenic chloride secretion in gut epithelia of normal and heterozygous CF mice. 2. Chloride secretory responses of normal and heterozygous colonic epithelia to forskolin, vasoactive intestinal polypeptide (VIP), isoprenaline, cholera toxin, heat-stable enterotoxin (STa), guanylin, carbachol and lysylbradykinin were examined. No significant differences in responses of tissues of the two genotypes were found. 3. Responses of normal and heterozygous ileal epithelia to forskolin and glucose were investigated. Heterozygous tissues responded as well as normal tissues. 4. Frusemide (furosemide) caused virtually identical inhibition of the chloride secretory responses to forskolin in colonic epithelia of both genotypes. 5. No evidence to support the genetic advantage hypothesis in ileal or colonic epithelia of the null CF mouse has been found, at least for acute responses. If the hypothesis is true then either (a) other non-cystic fibrosis transmembrane conductance regulator (non-CFTR) transport processes are involved, (b) prolonged exposure to secretagogues is required, or (c) delta F508 CFTR is responsible for the protective effect.

Topics: Animals; Bacterial Toxins; Calcium; Carbachol; Chloride Channels; Cholera Toxin; Colforsin; Colon; Cyclic AMP; Cystic Fibrosis; Enterotoxins; Epithelium; Escherichia coli Proteins; Furosemide; Gastrointestinal Hormones; Glucose; Heterozygote; Ileum; Isoproterenol; Kallidin; Mice; Mice, Mutant Strains; Natriuretic Peptides; Peptides; Vasoactive Intestinal Peptide

1. Cultured epithelia derived from whole human sweat glands, isolated secretory coils, isolated reabsorptive ducts and whole glands from cystic fibrosis (CF) subjects have been used to examine drug sensitivity by use of short circuit current recording. 2. Short circuit current increases were observed with lysylbradykinin, carbachol and histamine in epithelia of different origins. All responses were due to stimulation of electrogenic sodium absorption, evidenced by the inhibition of these responses by amiloride. The latter also abolished the basal current. The terpenes, thapsigargin and forskolin had no effect on transport. 3. The stimulation of a sodium current by agonists was dependent upon calcium, responses being inhibited by lanthanum ions and EGTA. Further A23187 induced a sodium current. 4. Pronounced oscillations in the sodium currents were a feature of the responses, implying synchronous, regulated cell activity. 5. Forskolin produced a ten fold increase in adenylate cyclase activity. All agonists listed in 2 except forskolin caused an increase in intracellular calcium [Ca]i, [Ca]i responses in CF cells were not different from those of normal cells, except with thapsigargin where the responses were smaller. 6. It is concluded that in culture, cells develop ductal characteristics, whether derived from normal or CF glands, coils or ducts. An increase in [Ca]i followed by activation of calcium-sensitive potassium channels and apical membrane hyperpolarization may be the major mechanism for increasing sodium influx.

Topics: Adenylyl Cyclases; Amiloride; Barium; Calcium; Carbachol; Cells, Cultured; Colforsin; Cyclic AMP; Cystic Fibrosis; Epithelium; Humans; Kallidin; Lanthanum; Sodium Channels; Sweat Glands; Terpenes; Thapsigargin

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.

Topics: Calcium; Cells, Cultured; Cystic Fibrosis; Drug Synergism; Electrophysiology; Histamine; Humans; Intracellular Fluid; Ion Transport; Kallidin; Methacholine Chloride; Signal Transduction; Sweat Glands

Prostaglandin E1 caused a dose-related inhibition of sodium reabsorption in the rat parotid gland when injected by retrograde perfusion into the glandular ducts. The extent of inhibition ranged from 11.7 +/- 2.4% at a dose of 2.5 micrograms to 63.8 +/- 8.9% at a dose of 31.2 micrograms. Both phospholipase A2, an enzyme involved in prostaglandin synthesis, and arachidonic acid, a precursor of prostaglandins, also increased the Na+ concentration of parotid saliva in a dose-dependent fashion. With phospholipase A2 the inhibition ranged from 21.6 +/- 4.4% at a dose of 3 micrograms to 73.5 +/- 8.2% at a dose of 30 micrograms. With arachidonic acid, the degree of inhibition was 5.1 +/- 3.0% at a 10(-5) M dose and 57.7 +/- 10.2% at a dose of 10(-3) M. Lysine bradykinin (kallidin), a peptide present in salivary and other exocrine glands and their secretions, also caused a 30% inhibition of Na+ reabsorption when retroperfused at a concentration of 12.5 micrograms, as did kallikrein (176 micrograms) and trypsin (33.3 micrograms). These results indicate that prostaglandins and kinins can inhibit Na+ reabsorption in the rat parotid duct when present in the luminal side of the cells. Since they are normally present in exocrine glands and can presumably be secreted, they may have a role as luminal factors in the regulation of transductal transport of Na+. The possibility that they may be increased in the exocrine secretions of patients with cystic fibrosis and that they may act as the so-called cystic fibrosis "factors" is also raised by the findings of this study.

Topics: Absorption; Animals; Cystic Fibrosis; Kallidin; Kallikreins; Male; Parotid Gland; Prostaglandins E; Rats; Rats, Inbred Strains; Sodium; Trypsin