John Atherton Young

Modification of salivary duct electrolyte transport in rat and rabbit by physalaemin, VIP, GIP and other enterohormones

The effects of various polypeptide enterohormones and the tachykinin secretogogue, physalaemin, on electrolyte transport by the main excretory duct of the mandibular gland of the rabbit were studied in vitro. Vasoactive intestinal peptide (VIP, 2×10−11 mol l−1) and gastric inhibitory polypeptide (GIP, 10−11 mol l−1) reduced nett Na+ movement from lumen to interstitium and VIP also reduced the transepithelial potential difference; the effective concentrations of the two hormones lay within the range of normal plasma concentrations. Gastrin (5×10−7 mol l−1) and synthetic secretin (2×10−7 mol l−1) had similar effects but only at concentrations well above the normal plasma levels. Caerulein, an analogue of the octapeptide of cholecystokinin, had no effect on duct function even at a concentration of 10−6 mol l−1. The potent salivary secretogogue, physalaemin (4×10−8 mol l−1), which is an analogue of SubstanceP, a putative mammalian enterohormone and neurotransmitter substance, caused a marked increase in ductal Na transport (in rat as well as rabbit). It is concluded that VIP and GIP would normally play a role in determining salivary electrolyte composition and it is postulated that their action may be antagonized by a tachykinin such as SubstanceP.

Microperfusion investigation of chloride fluxes across the epithelium of the main excretory duct of the rat submaxillary gland

ZusammenfassungNettochloridflüsse zwischen Interstitium und Lumen des Hauptganges der Glandula submaxillaris der Ratte wurden mit Hilfe der Mikrodurchströmungstechnik untersucht. Die Durchströmungsraten waren mit den unstimulierten Sekretionsraten der Speicheldrüse vergleichbar. Die Perfusionsflüssigkeit enthielt Inulin-14C und Gleichgewichtskonzentrationen von Natrium (2 bis 2,5 mÄq/l) und Kalium (125–135 mÄq/l). Als Anionen wurden entweder Chlorionen allein, Bicarbonationen allein oder 74 mÄq/l Chlor-und 63.5 mÄq/l Bicarbonationen verwandt.Bei der Durchströmung mit der Flüssigkeit, die nur Chlorid als Anion enthielt, gab es einen Nettochloridausstrom (von dem Lumen zum Interstitium), der bei schnellen Durchströmungsraten am größten war und sich bei kleineren Raten (verlängerter Durchflußzeit) dem Nullpunkt näherte. Mit der Perfusionsflüssigkeit, die nur Bicarbonat enthielt, gab es einen Nettochlorideinstrom. Auch dieser war bei schnellen Durchströmungsraten am größten und näherte sich bei kleineren dem Nullpunkt. Mit der Chloridbicarbonatflüssigkeit gab es nur einen kleinen Chlorideinstrom, der bei kleinsten Perfusionsraten (etwa 4 min Durchflußzeits) fast an den Nullpunkt kam. Die Chloridgleichgewichtskonzentration war 78,5 mÄq/l±1,5 (S.E.). Die interstitielle Chlorkonzentration war 118,0±1,1 mÄq/l.Aufgrund der bekannten Spannungsdifferenz (−11 mV, Lumen negativ) zwischen Lumen und Interstitium zeigen unsere Ergebnisse, daß Chlorionen zwischen Lumen und Interstitium passiv transportiert werden. Da Bicarbonationen entgegen dem elektrochemischen Gefälle in das Lumen transportiert werden, muß geschlossen werden, daß das Bicarbonatsystem (HCO3−, H+ bzw. OH−) mit einem aktiven Transportmechanismus gekoppelt ist.SummaryNett chloride fluxes between interstitium and the lumen of the rat submaxillary main duct were studied by microperfusion at constant rates comparable to unstimulated salivary flow rates. The perfusion fluid contained inulin-14C, and steady-state concentrations of sodium (2.5 mEq/litre) and potassium (125–135 mEq/litre); the anion content was either all chloride, all bicarbonate or a chloridebicarbonate mixture.During all-chloride perfusion there was nett chlorideefflux from lumen to interstitium which was greatest at high perfusion rates and fell towards zero as perfusion rate decreased (and contact time increased). During all-bicarbonate perfusion there was nett chlorideinflux, greatest at faster rates and falling towards zero as perfusion rate declined. Perfusion with chloride-bicarbonate mixture producted a small nett chloride influx at higher perfusion rates which fell almost to zero below 213 nanolitres/minute (contact time about 4 minutes). The steady-state luminal chloride concentration was 78.5 mEq/litre ± 1.5 (S.E.), and the mean interstitial chloride concentration was 118.0±1.1 mEq/litre.In conjunction with the previously determined steady-state trans-epithelial potential difference (−11 mV, lumen negative), these data show that chloride is passively distributed across the duct epithelium. Since preliminary data suggest that bicarbonate may be transported into the lumen against an electrochemical gradient, it seems probable that the bicarbonate system (HCO3−, H+, and OH−) is coupled in some way to an active transport mechanism.

Roles of the C Termini of α-, β-, and γ-Subunits of Epithelial Na+ Channels (ENaC) in Regulating ENaC and Mediating Its Inhibition by Cytosolic Na+

The amiloride-sensitive epithelial Na+ channels (ENaC) in the intralobular duct cells of mouse mandibular glands are inhibited by the ubiquitin-protein ligase, Nedd4, which is activated by increased intracellular Na+. In this study we have used whole-cell patch clamp methods in mouse mandibular duct cells to investigate the role of the C termini of the α-, β-, and γ-subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the β- and γ-subunits, but not the α-subunit, inhibited the activity of the Na+ channels. This mechanism did not involve Nedd4 and probably resulted from the exogenous C termini interfering competitively with the protein-protein interactions that keep the channels active. In the case of the C terminus of mouse β-ENaC, the interacting motif included βSer631, βAsp632, and βSer633. In the C terminus of mouse γ-ENaC, it included γSer640. Once these motifs were deleted, we were able to use the C termini of β- and γ-ENaC to prevent Nedd4-mediated down-regulation of Na+ channel activity. The C terminus of the α-subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na+ channels. We conclude that mouse Nedd4 interacts with the β- and γ-subunits of ENaC.

Patch-clamp studies on epithelial sodium channels in salivary duct cells

Mouse mandibular salivary duct cells contain an amiloride-sensitive Na+ current and express all three subunits of the epithelial Na+ channel, ENaC. This amiloride-sensitive Na+ current is subject to feedback regulation by intracellular Na+ and we have previously demonstrated that this regulation is mediated by an ubiquitin-protein ligase, which we identified as Nedd4. The evidence supporting this identification is as follows: (1) antibodies raised against murine Nedd4 block Na+ feedback inhibition; (2) a mutant of murine Nedd4 containing the WW domains but no HECT domain (ubiquitin-protein ligase) blocks Na+ feedback inhibition; and (3) Nedd4 is expressed in mouse mandibular salivary duct cells.In the present studies, we have used whole-cell patch-clamp methods to further investigate the mechanisms by which ubiquitin-protein ligases regulate the amiloride-sensitive Na+ conductance in mouse salivary duct cells. In particular, we have examined the possibility that the ubiquitin-protein which ubiquitin-protein ligases inhibit Na+ channels.We have found that KIAA0439 is expressed in mouse mandibular ducts and interacts with the PY motifs of the α-, β-, and γ-subunits of ENaC in vitro. Furthermore, in whole-cell patch-clamp studies, a glutathione-S-transferase (GST)-fusion protein containing the WW motifs of human KIAA0439 was able to inhibit feedback regulation of the amiloride-sensitive Na+ current by intracellular Na+. We also examined whether GST-fusion proteins containing the C-termini of the α-, β-, and γ-subunits of ENaC are able to interrupt Na+ feedback regulation of the amiloride-sensitive Na+ current. We found that the C-termini of the β- and γ-subunits were able to do so, whereas the C-terminus of the α-subunit was not.We conclude that KIAA0439 is, together with Nedd4, a potential mediator of the control of epithelial Na+ channels in salivary duct cells by intracellular Na+. We further conclude that ubiquitin-protein ligases interact with the Na+ channels through the C-termini of the β- and γ-subunits of the Na+ channels.

A micropuncture investigation of the cation content and osmolality of colloid from single rat thyroid acini

ZusammenfassungMit Hilfe der Nierenmikropunktionstechnik wurden Kolloidproben aus Acini der Schilddrüse von Ratten gewonnen und auf Osmolalität, Natrium-und Kaliumkonzentrationen untersucht. Es wurde folgendes gefunden: I. Das Follikelsekret war hyperton und hatte im Mittel eine Osmolalität von 323 mOsm/kg H2O; d.h. einen intrafollikularen hydrostatischen Druck von 365 mm Hg. II. Die Kaliumkonzentration des Sekrets (im Mittel 15,6 mÄq/liter) war dreimal so groß wie Plasma. III. Die Natriumkonzentration (im Mittel 160 mÄq/ liter) war gleich wie Plasma.Auf Grund der bekannten Spannungsdifferenz (0 bis −4 mV, Lumen negativ) zwischen Lumen und Interstitium zeigen unsere Ergebnisse, daß Kaliumionen vom Interstitium zum Lumen aktiv transportiert werden.SummaryRenal micropuncture techniques have been used to study the composition of the follicular secretion from single thyroid acini in the rat. Analysis of the secretion showed: I. The follicle contents were maintained hyperosmotic to plasma by about 21 mOsm/kg H2O, representing a hydrostatic pressure of about 365 mm Hg. II. The mean potassium concentration (15.6 mEq/litre) was significantly greater than in plasma. III. The mean sodium concentration (160 mEq/litre) was similar to that in plasma but showed a very wide scatter.Potassium appeared to have been concentrated in the lumen against an electro chemical gradient, since it is known that the trans-epithelial potential difference in the rat thyroid acinus is zero.

Kinetics of Na transport in the rat submaxillary main duct perfusedin vitro

SummaryThe main duct of the rat submaxillary gland has been isolated and perfusedin vitro while incubated in a plasma bath. It reabsorbs Na and secretes K and HCO3 at rates comparable to those reported for the ductin vivo and develops a transepithelial potential difference with magnitude dependent on the luminal Na concentration.The kinetics of ductal Na transport were studied by varying the Na concentration in the plasma bath step-wise from 149 to 320 mM and then the Na concentration in the luminal perfusate so as to produce zero nett flux of Na and water between lumen and bath. It was found that active Na transport by the duct showed saturation kinetics with a half-saturation constant of about 29 mM. The saturation curve could not be described in terms of a single Michaelis-Menten hyperbola although a more complex expression consisting of two such hyperbolic transport terms could easily be fitted to the data.Utilizing published values for unidirectional Na flux ratios it was calculated that the active Na transport rate when the luminal fluid had a plasma-like Na concentration was 4.37×10−9 mol·cm−2·s−1 and the epithelial Na permeability coefficient was 5.27×10−6cm·s−1.Although the kinetics of K and HCO3 secretion were not studied in detail it was observed incidentally that ductal secretion of these two ions still persisted even when their luminal concentrations were raised, respectively, to 197–225 mM and 80–120 mM.

Micropuncture and cannulation study of water and electrolyte excretion in the isotonic-secreting cat sublingual salivary gland

SummaryThe electrolyte composition of saliva from the isotonic-secreting cat sublingual gland has been compared with that of samples of acinar secretion obtained by micropuncture. The electrolyte concentrations in the acinar fluid, which were not affected by parasympathomimetic stimulation, resembled those of the primary fluid of hypotonic-secreting glands; they were [Na]=141 mM±5 S.E.M.; [K]=9.1±1.5 mM; [Cl]=128±4 mM.Following stimulation with carbachol, Na and K concentrations in final saliva showed, in contrast to saliva from hypotonic-secreting glands, only a slight flow dependence and Na concentrations were never lower, nor K concentrations higher than their respective concentrations in acinar fluid. Cl concentrations, on the other hand, were always greater than in primary fluid, ranging from 136±3 mM at low secretory rates to 162±6 mM at highest secretory rates.It was concluded that cat sublingual saliva is formed in 2 stages. First, an acinar secretion of approximately plasma-like electrolyte composition is formed by processes which seem essentially similar to those of all other exocrine glands so far studied by micropuncture. Second, the saliva is modified during passage along the gland excurrent duct system by secretion of Cl in exchange for HCO3. The duct system is atypical of salivary glands but seems to resemble that of the pancreas in lacking transport mechanisms for Na and K.

Development of glomerular filtration rate and electrolyte and osmolal clearance in the late-embryonic and newly hatched chicken

SummaryTechniques have been developed for collection of urine in embryonic and newly hatched chickens for the purpose of studying the development of renal function.The reliability of EDTA-51Cr as a substitute for inulin-14C in the determination of GFR was studied. Since inulin and EDTA-51Cr clearances in the hatched chicken averaged 1.61±0.23 (S.E.) ml/min per kg body weight and 1.58±0.27 ml/min per kg body weight, respectively, EDTA-51Cr clearance was considered a suitable measure of GFR.GFR increased significantly in the first few days after hatching. Filtration rate was 0.068±0.008 (S.E.) ml/min per g kidney weight in the embryo and increased to 0.148±0.008 ml/min per g shortly after hatching. By nine days after hatching GFR had risen to 0.290±0.015 ml/min per g, a value comparable to that reported for the adult.Clearances of sodium, potassium, chloride and total osmolyte also increased with age. When these clearances were corrected for changing glomerular filtration rates the embryonic chicks were found to excrete a greater percentage of the filtered load. These results show that adult levels of glomerulo-tubular balance are not attained until after hatching.

The action of physalaemin on electrolyte secretion by the mandibular and sublingual salivary glands of the rat@@@Der Einflu von Physalaemin auf die Elektrolyt-Sekretion der Submandibularis- und Sublingualisdrse bei der Ratte

ZusammenfassungDer Einfluß von Physalaemin und Acetylcholin auf den Wasser- und Elektrolyttransport wurde am Beispiel der Submandibularis- und Sublingualisdrüse der Ratte untersucht.Nach Stimulation mit Physalaemin fanden sich signifikant niedrigere Na+- und K+-Werte als vergleichsweise im acetylcholinstimulierten Speichel. Diese Befunde stimmen mit vorausgegangenen Mikroperfusionsstudien an der Submandibularis der Ratte überein. Letztere demonstrierten eine Stimulation der Na+-Reabsorption und Hemmung der Kaliumsekretion durch Physalaemin, während Acetylcholin einen entgegengesetzten Effekt zeigte.SummaryThe effects of physalaemin and acetylcholine on fluid and electrolyte excretion by the mandibular and sublingual glands of the rat were examined. Physalaemin-evoked mandibular and sublingual saliva was found to be poorer in sodium and potassium than acetylcholine-evoked saliva. These findings agree well with previous findings in the isolated perfused main excretory duct where physalaemin stimulates sodium reabsorption and inhibits potassium secretion, whereas acetylcholine has the opposite effect.