Ion Transport Responses Research Articles

The Small Intestine: Prospects for Therapeutic Approaches in Secretory Diarrhoeal Diseases

In many diarrhoeal diseases the intestinal mucosa is stimulated to secrete salt and water. This occurs in diarrhoea as a result of several bacterial toxins, is associated with inflammation and release of inflammatory mediators, and, in many instances, occurs when a neural element is evident. An understanding of the basic underlying mechanisms of secretion could lead to improvements in therapy. Development of vaccines against cholera is showing promise, and a knowledge of the complex field of inflammatory mediators, many of which provoke secretion, provides a foundation for development of more specific and selective anti-inflammatory agents. A detailed understanding of the complicated intracellular second-messenger systems, which are switched on by externally perceived signals, and of the ion transport responses, which are responsible for secretion, may lead to the development of specific anti-diarrhoeal drugs. Meanwhile, the message that oral rehydration therapy for severe diarrhoea, including cholera, is successful, should continue to be widely promulgated and taken up.

Depletion of guanine nucleotides with mycophenolic acid suppresses IgE receptor-mediated degranulation in rat basophilic leukemia cells.

In RBL-2H3 rat basophilic leukemia cells, Ag that crosslink IgE-receptor complexes stimulate the turnover of inositol phospholipids, the mobilization of Ca2+ from intra- and extracellular sources, the release of serotonin and other substances from granules and the transformation of the cell surface from a microvillous to a lamellar architecture. This study explores the role of GTP-binding proteins (G proteins) in the control of these biochemical and functional responses. We report that incubating RBL-2H3 cells for 4 h with 10 microM mycophenolic acid (MPA), an inhibitor of de novo GTP synthesis, reduces GTP levels by over 60% and causes an average reduction of 50% in Ag-stimulated serotonin release. This inhibition of secretion is associated with a 50% decrease in the rate of 45Ca2+ influx in MPA-treated cells. In contrast, Ag-stimulated inositol trisphosphate production is only slightly reduced, indicating that the phosphatidylinositol-specific phospholipase C can be activated by Ag in GTP-depleted cells. The membrane responses to IgE receptor cross-linking are unaffected by incubating cells with MPA. Exogenous guanine or guanosine protects the GTP pools in MPA-treated cells and permits normal ion transport and secretory responses to Ag; adenine does not. These results implicate a guanine nucleotide-binding protein in the control of IgE receptor-dependent signal transduction in RBL-2H3 cells. This protein may particularly control the Ca2+ influx pathway that is essential for secretion.

Electrophysiological responses of osteoclasts to hormones.

Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.

Primary culture of duck salt gland. II. Neurohormonal stimulation of active transport.

Primary cultures of structurally polarized sheets of avian salt gland secretory cells were mounted in Lucite chambers for transmural electrophysiological analysis. Transmural resistance values increased during the first 3 days of culture to 293 +/- 35 omega X cm2 and then decreased slowly thereafter. There was little short-circuit current (Isc) in the absence of secretagogues. Serosal addition of either carbachol or epinephrine resulted in a Isc consistent with positive charge flow from mucosa to serosa, thus demonstrating that these cell layers were capable of active ion transport in response to either cholinergic or adrenergic neurohormonal stimulation. Serosal ouabain or furosemide abolished the response to either agonist, while theophylline enhanced the response. Receptor specificity for the electrical responses was shown by selective inhibition of carbachol- and epinephrine-induced Isc by atropine and propranolol, respectively. The results demonstrate that these primary epithelial cell cultures are capable of active ion transport and are sensitive to known inhibitors of secretory transport, and suggest that intracellular coupling mechanisms for hormonal control are retained in culture. These cultures should be useful for studying mechanisms of ion secretory transport and their regulatory control.

Chymotrypsin, ileal chloride transport, and neurotransmitters.

Electrical field stimulation (EFS) depolarizes nerves and causes chloride secretion by mucosa of rabbit ileum mounted in a flux chamber. To test the hypothesis that the transmitter is a peptide, we determined whether the EFS response was prevented by the endopeptidase chymotrypsin (CT). Serosal, but not mucosal, addition of CT (200 micrograms/ml) reduced the short-circuit current (Isc) response to EFS by 90% or more. CT also reduced Cl absorption by decreasing the mucosal-to-serosal flux, but it did not affect net Na absorption. CT prevented the response to vasoactive intestinal polypeptides, but the response returned when CT activity was eliminated. The response to EFS did not return, however, implying that CT damaged cells that released transmitter or epithelial target cells. CT reduced the Isc response to serotonin by 69% and to A23187 by 10% and did not affect the theophylline response. We conclude that 1) the effects of CT on cell function limit its usefulness in identifying peptide neurotransmitters in epithelium, 2) CT irreversibly inhibits ion transport responses to EFS and to serotonin, and 3) CT reduces absorption of Cl probably by affecting a calcium pathway that modifies Cl transport.

Effect of theophylline and heat-stable enterotoxin of Escherichia coli on transcellular and paracellular ion movement across isolated porcine colon.

The effect of theophylline and a heat-stable enterotoxin of Escherichia coli (ST) on ion transport was examined using an in vitro, short-circuited preparation of the porcine colon. Theophylline abolished net Na absorption and elicited net Cl secretion, which quantitatively accounted for the increase in short-circuit current (Isc) observed. In contrast, a maximal dose of ST elicited an Isc response about one-half that of theophylline and only partially reduced the net absorption of Na and Cl. A significant residual ion flux, consistent with HCO3 secretion, was elicited by ST and was sustained after theophylline addition. Ion replacement experiments showed that the Isc and net ion transport response to ST was abolished when either Cl or HCO3 were removed from the bathing solutions. Voltage clamp experiments to evaluate the contribution of the paracellular and transcellular transepithelial pathways from serosa to mucosa showed that approximately one-half of the total serosa-to-mucosa flux (Jsm) of both Na and Cl was through the cells. Theophylline and ST both significantly reduced transcellular JNasm, but did not affect JClsm. Theophylline, but not ST, caused an increase in paracellular conductance of both ions. These results demonstrate significant differences in the effects of ST or theophylline on both transcellular and paracellular ion movement, and suggest that ST induces a Cl-dependent HCO3 secretion which is unobserved under control or theophylline-stimulated conditions. In addition, results are consistent with the operation of a neutral NaCl secretory process which is normally masked by the greater net rates of the neutral Na and Cl absorptive mechanisms. Thus, both ST and theophylline appear to reduce or abolished the neutral processes and convert the neutral secretory process into an electrogenic one. This latter effect could be explained simply by an increase in the anion conductance of the mucosal membranes.

Insulin stimulation of Na+ transport and glucose metabolism in cultured kidney cells.

A line of toad kidney cells (A6) in continuous culture was evaluated for ion transport and metabolic responses to insulin. The cells were grown on permeable supports to allow access of the medium to both basolateral and apical sides of the epithelium. Insulin, on the basolateral side only, produced an increase in short-circuit current (Isc) that was maximal at 40-60 min. A concentration-dependent increase in Isc and potential difference (PD) was found in the range of 10-3.2 X 10(3) microunits/ml insulin. The maximal stimulation of Isc and PD was approximately six- and twofold, respectively. After insulin exposure Isc was equivalent to net Na+ transport, indicating active Na+ transport stimulation. Insulin was also found to increase the incorporation of radiolabeled glucose into glycogen. Thus A6 cells exhibit both transepithelial transport and metabolic responses to insulin.

Influence of opiates on ion transport across rabbit ileal mucosa

Opiates are commonly used as antidiarrheal agents, and endogenous opioids have been demonstrated in the intestine. It seemed important therefore to investigate the effects of morphine on ion transport across intestinal mucosa. In rabbit ileum in vitro morphine (2 × 10− M) induced a significant fall in potential difference and short circuit current but did not influence tissue resistance. Dextromoramide (10−5 M), an active opiate, mimicked the action of morphine, whereas the inactive isomer levomoramide (10−5 M) had no effect. Morphine caused a significant increase in chloride absorption, due predominantly to a decrease in the serosa to mucosa flux. No change in sodium transport was detected, but the residual ion flux, possibly representing bicarbonate secretion, was enhanced. Similar responses were observed with a synthetic enkephalin analogue (Me-Tyr-D-Met-Gly-Phe-Pro-NH2); but this was more potent than morphine, a significant electrical response being observed at a concentration as low as 10−8 M. These electrical and ion transport responses to morphine were blocked by naloxone, an effect shown to be competitive in nature. The results suggest that opiate receptors exist in rabbit ileal mucosa and that these influence electrical and ion transport changes across the mucosa.

Theophylline-induced changes in ion transport and conductance in human small intestinal mucosa

THE central role of cyclic 3′,5′-adenosine monophosphate in intestinal secretion has been emphasised recently1,2. Electrolyte absorption is converted to secretion3,4 and mucosal conductance is reduced5,6 by increases in epithelial cyclic AMP concentrations and this is held to mediate the profound secretion provoked by cholera and other forms of infective ‘toxigenic’ diarrhoea7. The mechanisms of the intestinal response to cyclic AMP are incompletely understood and we therefore examined this response in human intestinal mucosa. We report here that changes in mucosal permeability are largely determined by the state of the lateral intercellular spaces. When these spaces are open, permeability is increased by cyclic AMP and when they are closed, permeability is decreased. These changes in permeability have a profound effect on the ion transport responses to cyclic AMP.

In vitro behavior of human intestinal mucosa. The influence of acetyl choline on ion transport.

The possibility that the autonomic nervous system may influence the function of intestinal mucosa was investigated by assessing the effect of acetyl choline on ion transport in human intestine. Isolated pieces of stripped ileal mucosa were mounted in Perspex flux-chambers and bathed in isotonic glucose Ringer's solution. Acetyl choline caused a rise in mean potential difference (8.8-12.3 mV, P less than 0.002) and short circuit current (287.7-417.2 muA-cm-2, P less than 0.01) (n = 12), observable at a concentration of 0.01 mM and maximal at 0.1 mM. This effect was enhanced by neostigmine and blocked by atropine. Isotopic flux determinations revealed a change from a small mean net Cl absorption (58) to a net Cl secretion (-4.3mueq-cm-2-h-1P less than 0.001) due predominantly to an increase in the serosal to mucosal unidirectional flux of Cl (10.63-14.35 mueq-cm-2-h-1P less than 0.05) and a smaller reduction in the mucosal to serosal flux (11.22 to 10.02 mueq-cm-2-h-1P less than 0.05). Unidirectional and net Na transport was unaffected. A similar electrical and ion transport response was observed in a single study of two pieces of jejunal mucosa. In the absence of glucose net chloride secretion was produced and again an insignificant effect on net sodium transport was noted. Acetyl choline did not provoke a sustained effect on mucosal cyclic adenine nucleotide levels although a short-lived cyclic adenine nucleotide response was seen in some tissues 20-30 s after drug addition. These studies demonstrate that acetyl choline does influence human intestinal ion transport by stimulating chloride secretion and suggest a possible mechanism by which the parasympathetic nervous system could be concerned in the control of ion transport.

Depressed Na-K-ATPase activity in the failing rabbit heart.

We studied the changes in the membrane ATPase from hypertrophied rabbit myocardium, and demonstrated a correlation between heart failure and ionic transport mechanism.In 17 animals cardiac hypertrophy was produced by constriction of the ascending aorta. A modification of the method of Tashima was used for preparation of membrane vesicles from the myocardium of the left ventricle. This preparation was carried out 10 days after the operation. Mgand Na-K-ATPases of the membrane ATPases were measured separately by their different responses to ouabain and cations.(1) Ventricular weight and heart weight were significantly greater in the experimental group than in the control group. However, the myocardial water content was almost constant in these groups.(2) The specific activity of Na-K-ATPase in myocardial vesicles was relatively high compared with skeletal muscle and corresponded to about one fourth of its total ATPase activity. This Na-K-ATPase activity was inversely correlated with the left ventricular weight (p<0.02). The difference in Na-K-ATPase activity between heart failure cases and controls was significant at the 5% level. On the other hand, no significant difference in Mg-ATPase activity between the failure and the control group was demonstrated.These results supported the fact that, during the development of heart failure, the myocardium lost potassium. From these findings, it was postulated that the heart in failure would be characterized by a diminished ability to manipulate active ion transport in response to hemodynamic stress due to the decrease in Na-K-ATPase activity.

Parathyroid Hormone-induced Mitochondrial Swelling

Abstract In addition to stimulating ion transport and respiration, it is now established that the parathyroid hormone induces rapid and extensive mitochondrial swelling. The swelling process takes place in the absence of Mg++ in a medium composed of sucrose, substrate, and tris(hydroxymethyl)aminomethane. If Mg++ is present, the swelling change is greatly minimized. The swelling is accompanied by a simultaneous increase in pyridine nucleotide oxidation and respiration, and is neither dependent upon nor enhanced by K+ or Na+. Inorganic phosphate, acetate, and, to a lesser extent, arsenate stimulate the rapidity and extent of the parathyroid hormone-induced swelling. The results suggest that parathyroid hormone-induced swelling is closely related to the ion transport and respiration responses.