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Kinetics of bidirectional active sodium fluxes in the toad bladder.

The kinetics of isotopic Na+ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal 22Na+ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na+ flow. The electrical current I and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at deltapsi=0, 25, 50, 75 or 100 mV. Subsequent elimination of active Na+ transport with mucosal amiloride permitted calculation of the rates of active Na+ transport JaNa and active and passive influx leads to JaNa and leads to JpNa. The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence JaNa=Ja. Ja was abolished at deltapsi=E=96.3+/-1.9 (S.E.) mV. As deltapsi approached E, active efflux comes from Ja became demonstrable. At deltapsi=100 mV, comes from Ja exceeded leads to Ja, so that Ja was negative. Experimental values of leads to Ja agreed well with theoretical values predicted by a thermodynamic formulation: leads to Jaexp=0.985 leads to Jatheor (r=0.993). The dependence of leads to Ja on deltapsi is curvilinear.

Amiloride↗

Alterations in electrophysiology of isolated amphibian small intestine produced by removing the muscle layers.

Isolated segments of Amphiuma small intestine bathed in chloride or sulfate buffer generate a greater short-circuit current and a larger change in current in response to galactose when the serosal muscle layers are stripped from the mucosa. Intact (unstripped) segments are not apparently anoxic since stripped segments exposed to serosal N2 for 3 h display normal short-circuit currents but a reduced potential response to galactose, while the presence of muscle layers tends to reduce the short-circuit current but does not alter the potential response to galactose. Bullfrog small intestine also generates greater short-circuit current following removal of the muscle layers. The enhancing effect of stripping appears to be related to removal of a resistance to ion flow across the tissue.

Amphibians↗

Does intracellular sodium regulate sodium transport across the mucosal surface of frog skin?

A method has been devised to functionally remove the serosal membrane of frog skin. Skins treated in this way have no spontaneous potential. However, if sodium gradients are placed across the tissues diffusion potentials and hence short-circuit currents of either sign, depending on the direction of the gradient, could be recorded. These short-circuit currents were completely imhibited by amiloride only from the mucosal face. However, the concentration of amiloride causing 50% inhibition of the short-circuit curent (Km) in treated skins was 2.3 . 10(-3)M, when a sodium gradient was applied from serosa to mucosa, whereas both in untreated skins without a sodium gradient and in treated skins with a mucosal to serosal sodium gradient, the Km of amiloride was 2 . 10(-7)-4 . 10(-7)M. The mechanism by which amiloride is able to inhibit the short-circuit currents of either sign is discussed.

Amiloride↗

Localization and characterization of transport-related elements in the plasma membrane of turtle bladder epithelial cells.

A mixed membrane preparation obtained from turtle bladder epithelial cells contains (Na+ + K+)-ATPase, adenylate cyclase and protein kinase, which interact with ouabain, norepinephrine and cyclic AMP, respectively. When such a preparation is obtained from bladders which had been preexposed to serosal fluids containing the tritiated form of 4,4'-diisothiocyano-2,2'-disulfonic stilbene, the subsequently isolated membrane proteins are enriched in tritium as well as in the afore-mentioned enzymes, none of which is inhibited. Free-flow electrophoresis separates the mixed membrane preparation into two distinguishable groups: one, construed as apical membranes, is enriched in norepinephrine-sensitive adenylate cyclase and cyclic AMP-sensitive protein kinase; the other, construed as basal-lateral membranes, is enriched in ouabain-sensitive ATPase and 4,4'-diisothiocyano-2,2'-disulfonic stilbene-binding proteins. The physiological counterparts of these enzymatically defined membrane markers are the mucosal sidedness of the transport effects of norepinephrine and cyclic AMP derivatives and the serosal sidedness of the transport effects of ouabain and disulfonic stilbenes in the intact turtle bladder. The discreteness and ion selectivity of each membrane-bound, transport-related element are discussed in relation to the corresponding characteristics of each transport process in vivo; the possibility of regulation of anion transport by adenylate cyclase-protein kinase system is also discussed.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

The regulation of volume and ion composition in frog skin.

1. Chemical determinations of Cl- in frog skin indicate that the intracellular concentration of this ion is above that corresponding to an equilibrium distribution. 2. Published data on Na+ efflux from the cell compartments into the two external bathing solutions, and on unidirectional fluxes across the whole preparation suggest that there are large unidirectional fluxes across the basolateral membranes. 3. Cl- uptake from the mucosal barrier is very small, and the removal of Cl- from the mucosal bathing solution does not affect the intracellular Cl- concentration. 4. Removal of Cl- from the serosal bathing solution produces a drastic decrease in cell Cl- together with a loss of water. 5. This is accompanied by a less marked effect on Na+ and K+ content of the cells. 6. The removal of Na+ from the serosal bathing solution produces also a decrease in Cl-, Na+ and K+ content of the cells with a marked loss of water. 7. It is suggested that the basolateral membrane of the frog skin is the site of mechanisms able to regulate volume and ion composition of the epithelial cells and that part of these mechanisms consists of a coupling between the movements of Na+ and Cl-.

Acetazolamide↗

Effect of 3-isobutyl-1-methylxanthine on HCO3- transport in turtle bladder. Evidence for electrogenic HCO3- secretion.

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO3-/CO2-containing, Cl- -free Na+ media exhibit a short-circuit current (Isc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the Isc and p.d. The IBMX-induced reversal of Isc and p.d. is (1) dependent on the presence of HCO3- (and CO2) in the serosal bathing fluid, (2) independent of Na+ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed Isc elicited by IBMX represents electrogenic secretion of HCO3-.

1-Methyl-3-isobutylxanthine↗

Reversal of copper-induced inhibition of vasopressin responsiveness by reducing agents.

Copper inhibits the hydro-osmotic response to vasopressin in the urinary bladder of Bufo marinus at a site proximal to cyclic AMP production. This effect is not reversed by washing in Cu2+ -free Ringer's solution but is overcome by serosal addition of reducing agents, suggesting that vasopressin responsiveness in this tissue is modulated by either the redox potential or the sulfhydryl content of the serosal membrane.

Animals↗

Effect of furosemide on ion transport in the turtle bladder: evidence for direct inhibition of active acid-base transport.

The diuretic furosemide inhibits acid-base transport in the short-circuited turtle bladder. It inhibits luminal acidification when present in either mucosal or serosal bathing fluids, but decreases alkalinization only from the serosal side of the tissue. The inhibition of both acid-base transport processes is independent of ambient Cl-; and the disulfonic stilbene, SITS, an inhibitor of Cl--HCO3- exchange, fails to prevent the furosemide-elicited inhibition of alkalinization. These results preclude an absolute requirement of a furosemide-sensitive Cl--HCO3- exchange by these transport processes. The drug also interferes with the CO2-induced stimulation of acidification and alkalinization. The inhibition of the residual acidification in acetazolamide-treated, acidotic bladders, however, suggests an action at sites other than cytosolic carbonic anhydrase. Although active Na+ and Cl- reabsorption and tissue oxygen uptake are also decreased by furosemide, the rate of oxygen consumption uncoupled by 2,4-dinitrophenol is not diminished, indicating a primary inhibition of the various ion transport processes, not of metabolism. It is proposed that inhibition of transepithelial acid-base transport by furosemide in the turtle bladder includes inhibition of the acid-base pumps.

2,4-Dinitrophenol↗

Phorbol myristate acetate induces endocytosis as well as exocytosis and hydroosmosis in toad urinary bladder.

The induction of the hydroosmotic response in the toad urinary bladder is considered to be associated with membrane addition mediated by exocytosis at the affected luminal membrane and reversed by endocytic retrieval at that surface. The permeability, exocytosis and endocytosis are initiated by antidiuretic hormone (ADH) receptor interaction on the basolateral membrane. In other hormone responsive systems, phorbol ester (phorbol myristate acetate, PMA), a tumor promoter, has been implicated in the regulation of various transport processes through the activation of protein kinase C and cytoskeletal protein phosphorylation. We found that addition of 10(-6) M PMA to the mucosa induces an hydroosmotic response which is gradual and which reaches a maximum within 60 min, equal to about 1/3 the maximal ADH response. Morphologically, PMA causes rapid exocytosis of the granules, endocytosis of horseradish peroxidase from the mucosal medium into tubules and multivesicular bodies and elongation of apical microvilli. Controls treated with mucosal 0.1% dimethylsulfoxide (DMSO) or an inactive PMA isomer on the mucosal surface, or PMA on the serosal surface lack the hydroosmotic, exocytic, endocytic and cytoskeletal changes. Addition of serosal ADH to PMA-treated bladders results in a precocious hydroosmotic and exocytic ADH response, but a lowering of the maximal response. Also pretreatment of bladders with PMA prevented the ADH-induced increase in transepithelial potential difference. Thus, apical events mediating the PMA hydroosmotic response are correlated with exo- and endocytosis and elongation of apical microvilli.

Animals↗

Calcium-dependence of sugar transport in rat small intestine.

The involvement of Ca2+ in the theophylline action on sugar transport was investigated in isolated rat small intestinal mucosa. Theophylline significantly increased cell water free sugar accumulation and reduced mucosal to serosal sugar fluxes both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium free media. In theophylline untreated tissues, calcium-deprived bathing solutions decreased tissue galactose accumulation and increased mucosal to serosal sugar flux. The calcium-channel blocker verapamil produced similar effects on intestinal galactose transport to those induced by low extracellular calcium activity. RMI 12330A and the calmodulin antagonist trifluoperazine abolished the theophylline-effects on intestinal galactose transport. Both drugs also affected sugar transport in basal conditions. These studies suggest that calcium might modulate sugar permeability across the basolateral boundary of rat enterocytes, and that its effect may be mediated by calmodulin.

Animals↗

Thiazides stimulate calcium absorption in urinary bladder of winter flounder.

Thiazides inhibit voltage-independent NaCl absorption in the urinary bladder of the winter flounder presumably by blocking an electroneutral mucosal Na/Cl co-transporter. As thiazides stimulate calcium absorption in mammalian distal convoluted tubule while inhibiting NaCl absorption, we studied the effects of hydrochlorothiazide (HCTZ) on unidirectional 45Ca fluxes and intracellular electrical potential in short-circuited bladders to examine possible mechanisms of HCTZ effects on calcium transport. Basal secretory calcium flux was, on average, slightly larger than absorptive flux, reflecting small net calcium secretion. Mucosal addition of HCTZ (10(-4) M) stimulated absorptive calcium flux by 46% while the secretory flux was unaltered. Thus, HCTZ tended to induce net calcium absorption. Pre-treatment with serosal ouabain (10(-4) M) attenuated the HCTZ-induced increase in absorptive calcium flux. Moreover, HCTZ hyperpolarized the mucosal membrane potential by 18% as measured by conventional open-tip microelectrodes. These effects of HCTZ are consistent with the hypothesis that HCTZ indirectly stimulates Na/Ca exchange located at the serosal membrane. In conclusion, HCTZ in flounder urinary bladder, as in mammalian distal convoluted tubule, simultaneously inhibits NaCl absorption and stimulates calcium absorption. This study expands on the functional similarities between the flounder urinary bladder and the mammalian distal convoluted tubule.

Animals↗

Highly selective blockade of the frog skin sodium channels by monovalent copper cations.

.5 mM Cu+ added to the mucosal side of frog skin caused rapid reversible inhibition of short-circuit current while no effect of Cu+ could be observed at the serosal side. In both cases Cu2+ was reduced to Cu+ by adding 10 mM ascorbic acid. Cu+ being similar to Na+ both in charge and crystal radius (0.096 and 0.095 nm, respectively) appears to block Na+ channels in the apical membrane. Cu2+ being of a smaller size (crystal radius 0.072 nm) was ineffective at the mucosal side causing only a rather slow irreversible inhibition of Na+ transport when added to the serosal bathing solution.

Animals↗

The action of aldosterone and ouabain on cation concentrations in incubated renal slices.

Studies have been made on the effects of aldosterone and ouabain on Na+ and K+ concentrations in incubated rat kidney slices. The results indicate that, control slices gained Na+ over the first 10 min of incubation and subsequently extruded it, with increase in incubation time. In the presence of aldosterone, the same pattern was observed as with control slices, but tissue Na+ concentrations were significantly higher at all times. In the presence of ouabain, slices accumulated Na+ continuously, but in the presence of both drugs, tissue Na+ concentrations were not as high as with ouabain alone. Both aldosterone and ouabain increased the loss of cell K+ whereas control slices reaccumulated their intracellular K+ concentration, with increase in incubation time. The results suggest that aldosterone may have an actual mechanism of action in the renal tubule: either by activating two Na+, K+ pumps located on the luminal and serosal membranes, respectively, or by enhancing luminal membrane permeability, and at the same time stimulating Na+, K+-ATPase on the serosal membrane.

Aldosterone↗

Stimulatory effect of angiotensin II on the electric properties of the isolated toad skin.

In 1982 we showed that angiotensin II (Agt II) stimulates the bioelectric properties of the isolated toad skin and that this effect is blocked by pretreatment of the skin with indomethacine [J. B. Concha et al., IRCS Med. Sci. 10, 584 (1982)]. Ussing's technique and several inhibitors were used to continue this study on the isolated Pleurodema thaul skin. Serosal Agt II produced a dose-dependent increase in electrical parameters: a maximal concentration of 6 X 10(-6) M Agt II increased potential difference by 43 +/- 7.8% and short-circuit current by 51.5 +/- 7.7%. The responses were not affected by either alpha or beta blockers or by atropine. Indomethacine blocked responses to the calcium ionophore A23187 and to Agt II which were similar to each other. Additive effects of Agt II and of the calcium ionophore A23187 were found. No response to Agt II was obtained when Ca2+-free Ringer was used on the serosal side. Calcium channel blockers (nifedipine, verapamil, manganese), pentobarbitone and saralasin blocked the response to Agt II. This pharmacological evidence is in favour of the hypothesis that Agt II activates specific membrane receptors, leading to Ca2+ release and formation of prostaglandins which stimulate adenyl cyclase. This increases cAMP secretion, which in turn increases apical membrane permeability to sodium and enhances the active transport system.

Adrenergic alpha-Antagonists↗

Absorption of N4-D-glucopyranosylsulphamethazine by rat everted intestinal sacs.

Absorption of the N4-D-glucose conjugate of sulphamethazine (glucose-SMZ, 0.5 mM) by isolated everted sacs of the rat small intestine was studied at 37 degrees and pH 6.6. Phlorizin (0.5-2.0 mM) significantly reduced (P < 0.05) both mucosal and serosal transfer of glucose-SMZ and inhibition of mucosal transfer appeared to be concentration-dependent. Phloretin (0.5 mM) and removal of Na+ from the incubation medium also diminished absorption of glucose-SMZ. Furthermore, D-glucose (0.5 and 5.0 mM) inhibited mucosal and serosal transfer of the glycoside. The results suggest the D-glucose/Na+ cotransporter mediates absorption of glucose-SMZ from the small intestine of the rat. Thus, glucose-SMZ might be bioavailable from ingested tissues in which it is present.

Animals↗

The role of mast cells in demyelination. 1. Myelin proteins are degraded by mast cell proteases and myelin basic protein and P2 can stimulate mast cell degranulation.

Incubation of bovine peripheral myelin with supernatants from degranulated rat serosal mast cells led to extensive loss of P0. Similarly, when myelinated axons prepared from guinea pig CNS were incubated with degranulation supernatants, a significant loss of basic protein (MBP) was observed. As cationic peptides can stimulate mast cell degranulation, rat serosal mast cells were incubated with MBP, and with P2. Degranulation was assayed by measurement of release of the granule enzyme beta-hexosaminidase and it was found that both MBP and P2 stimulated 40-50% degranulation at a concentration of 50 micrograms/ml. The results of this study suggest that release of mast cell proteases could contribute to myelin damage in both the PNS and CNS, and that subsequent release of P2 or MBP or their breakdown products could potentiate further mast cell degranulation.

Animals↗

Intestinal absorption of arsenate in the chick.

The intestinal absorption of arsenate(As(V)) has been investigated in the chick by means of the in situ ligated duodenal loop technique. By this procedure, it was observed that arsenate is rapidly and essentially completely absorbed (80-95%) from the lumen at As(V) concentrations up to 5 mM, declining to about 50% absorption at 50 mM. Transfer from the intestinal lumen to the mucosal cells at low As(V) concentration (0.1 mM) is rapid, while transfer from the mucosal cells to the body occurs more slowly. At stable As(V) concentrations greater than 1 mM, fractional mucosal cell accumulation of As(V) remains constant, while fractional transfer to the body declines. However, total mucosal accumulation of As(V) and that transferred to the body increase in a linear logarithmic fashion from 0.05 to 5 mm As(V). The results indicate that As(V) readily penetrates both the mucosal and serosal surfaces of the epithelial membrane. Furthermore, arsenate and phosphate do not appear to share a common transport pathway in the duodenum and no evidence was obtained for any interaction between the two at this level. Vitamin D3 administration to rachitic chicks was effective in significantly elevating duodenal arsenate absorption, acting primarily to enhance serosal transport.

Administration, Oral↗

Induction of guinea pig airway hyperresponsiveness by inactivation of guanylate cyclase.

To examine the role of cyclic 3', 5'-guanosine monophosphate (cGMP) in airway responsiveness the effects of substances known to interfere with nitric oxide (NO) or cGMP were investigated on guinea pig airways. Using a perfused organ bath system, it was possible to apply the chemicals from either the serosal or the mucosal side independently. In addition, levels of intracellular cGMP were determined in tissues after various treatments. Sodium nitroprusside (a donor of NO), zaprinast (a specific inhibitor of cGMP phosphodiesterase) and 8-bromo-cGMP (8-Br-cGMP) caused a concentration-dependent relaxation of guinea pig trachea. These results indicate that cGMP is an important second messenger mediating tracheal relaxations. The above mentioned drugs caused a more profound relaxation when applied to the serosal side compared to the mucosal side, suggesting a barrier function of the epithelial layer. Incubation on the mucosal side of the tissues with 100 microM pyrogallol (a generator of superoxide that may inactivate NO) increased the contractile response to histamine at concentrations 0.3-3.2 microM (P < 0.05). Treatment of the preparations with 1 mM cystamine (an inactivator of guanylate cyclase) caused a 5-fold increase in the sensitivity to histamine (P < 0.05), indicating the involvement of the NO/cGMP pathway in the development of airway hyperresponsiveness. Incubation of the tissues with 100 microM histamine elevated the intracellular cGMP levels 10-fold; this effect was completely prevented by incubation of the tissues with methylene blue (a potent inactivator of guanylate cyclase). Mucosal incubation of the tracheal tubes with 10 microM methylene blue induced an 8-fold increase in sensitivity to histamine (P < 0.01) and the Emax was slightly increased. 25 min after instillation of 0.4 mumol methylene blue into the airways of anaesthetized guinea pigs, the lung resistance in response to histamine was elevated up to 395 +/- 82% (P < 0.001). The present study revealed that inactivation of NO or guanylate cyclase enhances the histamine-induced contractions of guinea pig tracheas. Therefore, it is suggested that the NO/cGMP pathway may be implicated in the pathogenesis of airway hyperresponsiveness and that drugs which enhance cGMP levels in airway smooth muscle may be of significance in the treatment of airway obstruction and enhanced reactivity.

3',5'-Cyclic-GMP Phosphodiesterases↗