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Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested in Necturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 micrograms/ml = 20 microM), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (psi ms) turned from slightly mucosa-positive values to mucosa-negative values of approximately +1 to -5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 micrograms/ml but a maximum response was achieved between 10 and 20 micrograms/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5 X 10(-4) M) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability. [ABSTRACT FROM AUTHOR]

Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 microM), induces about a twofold increase in transepithelial resistance in Necturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where greater than 90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular action a priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50 mM) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixed in situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction. [ABSTRACT FROM AUTHOR]

A short-circuit current experiment on epithelial ion transport is described that is suitable for student classes in human and animal physiology. Segments of late distal colon from either pig or cow are obtained from the slaughterhouse depending on the animals' daily schedule. Initial tissue preparation already in the slaughterhouse, cold storage, and proper choice of bath solutions are essential prerequisites for success. Students monitor spontaneous transepithelial voltage and short-circuit current (Isc) by use of manually operated voltage clamp units. Two main transport mechanisms are studied, electrogenic Na+ absorption and Cl- secretion. Electrogenic Na+ absorption is studied by measuring the Isc drop after amiloride. Then Cl- secretion is stimulated by theophylline and subsequently inhibited by furosemide. In some experiments K+ secretion can be detected by the blocking effect of mucosal Ba2+. Response of tissues from pig and cow is qualitatively similar but quantitatively different. The equipment is sturdy and inexpensive, can be provided by most departmental workshops, and has been tested for 3 yr in regular lab courses. Observations made during these experiments are closely related to clinical states, such as secretory diarrhea, cystic fibrosis, and hyperaldosteronism, as well as to the mechanisms of clinically used diuretics.

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl- channel (ORDIC) during cAMP-mediated Cl- secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl- channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4'-dinitro-2,2'-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ODIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl- secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC50 = 0.5 +/- 0.3 mumol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl- secretion unless high concentrations were used (IC50 = 240 +/- 10 mumol/l). In order to exclude effects of NPPB other than on the apical Cl- channel, transepithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl- gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl- current showed a very-low-frequency Lorentzian noise component (fc = 1.4 +/- 0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC.(ABSTRACT TRUNCATED AT 250 WORDS)

Protamine reversibly decreases cation permeability and alters the structure of Necturus gallbladder tight junctions. Conflicting results, however, have been published whether or not it also affects apical cell membrane permeability. We investigated this issue more systematically by measuring voltage (psi mc) and fractional resistance (fRa) of the apical membrane at varying concentrations of protamine, K+, and H+ in the bathing solution. At pH 7.6 and [K+] 2.5 mM, (Poler, M.S. and Reuss, L. (1987) Am. J. Physiol. 253, C662) 6 microM protamine caused psi mc to depolarize from -58 to -51 mV and fRa to decrease from 0.74 to 0.67. If we increased pH to 8.1 these effects were even more pronounced. At [K+] 2.5 mM, but not 4.5 mM, psi mc transiently hyperpolarized for about 5 min after adding protamine. Most importantly, if [K+] was 4.5 mM and pH was adjusted to 7.1 (Bentzel et al. (1987) J. Membr. Biol. 95, 9) no significant changes of psi mc and fRa occurred. In any case, at a supramaximal concentration of 200 microM, protamine did not further increase the paracellular response but produced decreasing psi mc and fRa. We conclude that 6 microM protamine decreases K+ conductance of the apical membrane, if it is already tuned high by high pH. At low control K+ conductance as observed at lower pH, protamine action is restricted to the paracellular pathway. Thus, conflicting results were due to different experimental conditions. At a solution pH of 7.1, 6 microM protamine fulfills criteria of a selective tool for reversibly altering structure and function of the tight junction in Necturus gallbladder.

The short-term action of aldosterone in physiological concentration on net fluxes of Na, K, Cl, HCO3, osmolytes, and water was examined in the proximal colon and rectal colon of adrenalectomized (ADX) rats in vivo. The measuring time was 12 h, divided in eight periods of 90 min. (a) Aldosterone alone (6 nmol h-1 kg-1) did not stimulate transport in ADX rats. In these experiments plasma [K] increased to fatal values. A basal glucocorticoid substitution of 24 nmol h-1 kg-1 corticosterone caused plasma K to stay constant throughout the experiment, so that epithelial transport was not handicapped by non-specific effects of ADX, but this also did not restore the decreased transport of ADX rats to control values. Under these conditions (absence of aldosterone) in the rectal colon Na and H2O transport was zero, whereas in the proximal colon flux rates were depressed by between 30% and 50%. In contrast, basal glucocorticoid substitution of 18 nmol h-1 kg-1 corticosterone plus infusion of 6 nmol h-1 kg-1 aldosterone caused transport stimulation to values not significantly different from those of non-ADX controls. We conclude that after ADX, aldosterone at physiological concentrations increases transport if, as a prerequisite, a basal glucocorticoid substitution is provided. Transport of Na, K, and H2O is under the total control of aldosterone in the rectal colon but is only moderately altered in the proximal colon.(ABSTRACT TRUNCATED AT 250 WORDS)

Functionally isolated segments of rat colon and rectum were perfused in situ in a closed loop system. Rectum was defined as the lower 25--35% of the length of large intestine (cecum excluded). Perfusion conditions were optimized at 0.5 ml.min-1 and 3 cm H2O luminal pressure. Variation of perfusion rate between 0.2 and 2 ml.min-1 did not influence net volume transport (JNV). Luminal distension following elevation of hydrostatic pressure to 18 cm H2O reversibly increased Jnv. Under control conditions Jnv and Na+-transport rates (JnNa) of colon were 2--3 times higher than those of rectum. In colon transepithelial electrical potential difference (psims) was time independent --12 mV (lumen negative) whereas rectal psims increased with time from --6 mV, reaching a plateau of --67 mV within 6 h. Amiloride 10(-4) mol.l-1 had no effect on psims, Jnv, and JnNa in colon but did slightly depress K+-secretion in colon descendens. In contrast, psims in rectum was dose-dependently depressed, being reversed to +7 mV at 10(-4) mol.l-1. Jnv and JnNa were decreased by half. Acetazolamide in addition to amiloride lowered the positive post-amiloride rectal psims by half. Adrenalectomy had no effect on colonic psims, but abolished psims of the rectum. A single dose of 40 microgram.kg-1 b.w. aldosterone during the experiment restored the typical time course of rectal psims, but did not affect psims in colon. It is concluded that aldosterone induces an amiloride-sensitive Na+-pathway only in rectum, but not in colon, and that colon and rectum differ basically in their transport properties, quantitatively as well as qualitatively, as do the kidney distal convoluted tubule and the cortical collecting duct.

A simple and versatile tool facilitating micropuncture of small cells is described which utilizes a commercial piezoelectric element made from a stacked column of monomorph ceramic discs. The device is able to advance complete input stage-electrode-assemblies with high speed and can be used in combination with conventional micromanipulators. Advancing characteristics as recorded optically at high magnification demonstrated less axial vibration, although faster action, than two other modern micropositioners driven by step motors. In biological experiments on selected tissues (Necturus gallbladder epithelium, Amphiuma renal distal tubule cells, rabbit and human corneal endothelium) the combined use of micromanipulator and piezo-stepper was, in all cases, superior to the use of a micromanipulator alone: the percentage of successful cell penetrations increased, cell potentials were stable for a longer time, and the durability of electrode-tips improved.

The transepithelial voltage (psi ms) of rat rectum in vivo increases for several hours in experiments under general anaesthesia. So far this was attributed by indirect evidence to increasing aldosterone plasma levels during the course of the experiment. We performed direct measurements of aldosterone and corticosterone plasma concentrations during intestinal perfusion experiments on barbiturate anaesthetized rats. Experiments were terminated for blood sampling at 10, 75, 300, 400, 800, or 1,800 min, respectively. (i) After 75 min of anaesthesia, surgical preparation was finished and plasma levels of aldosterone and of corticosterone were found increased by the factors 5 and 3, respectively, as compared to conscious controls. (ii) During the following 12 h, aldosterone further increased to levels 10 times as high as those of controls. In contrast, during the same period corticosterone slowly decreased but still remained elevated as compared to controls. (iii) The increase of both hormones was attenuated when abdominal surgery was omitted. (iv) The use of pentobarbital (Nembutal) instead of thiobarbital (Inactin) did not influence the adrenal response. (v) In adrenalectomized rats a continuous substitution with 65 ng X h-1 X kg-1 BWT aldosterone resulted in plasma levels as high as in conscious intact animals. (vi) Rectal psi ms started to move to higher lumen-negative values with a time delay of 1-1 1/2 h as compared with the increase of hormone levels. psi ms then stayed elevated until to the end of the experiments. We conclude that in vivo experiments of several hours duration in thio- or pentobarbital anaesthetized rats take place under conditions of aldosterone and corticosterone plasma levels which are high as compared to those of conscious unstressed animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiobutabarbital anaesthetized and abdominally operated control rats develop high endogenous plasma levels of both aldosterone and corticosterone during the course of a 12 h experiment. This effect was used as a model for examining 'acute' steroid action (i) on net ion and water fluxes and (ii) on zero flux luminal limiting concentrations in rat upper colon (proximal 50% of large intestine) and rectum (distal 40%). Experiments of both kinds consisted of 8 independent 90 min measuring periods. (i) In rectum net fluxes of Na, K, osmolytes (sum of all solutes) and water started at low levels around zero, began to rise about 2 h after plasma levels of aldosterone had increased, and reached plateau values around the 6th hour of anaesthesia. In upper colon, fluxes of Na, K, Cl, and osmolytes were high from the beginning and did not vary significantly with time. (ii) At zero flux conditions limiting concentrations of Na in the hormonally unstimulated phase of the experiment were 20 +/- 3 mM in upper colon and 22 +/- 3 mM in rectum. After maximal endogenous aldosterone liberation zero flux concentrations were 5.2 mM in upper colon and 2.2 mM in rectum, corresponding to luminal fluid to plasma ratios (LF/P) of 0.040 and 0.016, respectively. Amiloride reduced the maximal Na gradient in rectum to a LF/P of 0.3 but was not effective in upper colon and did not prevent the stimulating effect of aldosterone in this segment. Under all experimental conditions zero flow concentrations of K were higher than consistent with a solely passive distribution, indicating simultaneous passive and active secretion in both segments. In contrast to the findings of others, the luminal fluid remained isoosmolar with plasma in all zero flux experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial and subepithelial electrical resistances of rat large intestine were measured by means of a 4-electrode AC impedance technique in three segments, colon ascendens, colon descendens and rectum. Epithelial resistance of colon ascendens and colon descendens was about 35 omega X cm2 and not different between these two segments. It was, however, about 3 times higher in rectum (99 omega X cm2). This finding is in accord with our previous observation of about 3-fold higher net fluxes of ions and water in colon ascendens and colon descendens than in rectum. It confirms the concept of a main functional difference between the terminal part of the large intestine (rectum) and the more proximal segments (colon). The acutely (within hours) varied level of aldosterone by keeping the rats for 7 h in anaesthesia caused in the rectum a more than 10-fold increase in short circuit current (Isc) and transepithelial voltage but no significant decrease in resistance. Similarly, the decline in Isc, as regularly observed in the early phase of in vitro measurements on partially stripped large intestine, was paralleled by voltage changes but not by changes in resistance. We conclude that the wide range of resistance values published so far was caused to a great extent by including various portions of colon or rectum. By comparing intact (not stripped) and partially stripped preparations (muscularis propria removed) of the rectum it was shown that partial stripping did not alter the epithelial resistance but reduced the subepithelial resistance in this segment from 26 to 8 omega X cm2, or by 68%.(ABSTRACT TRUNCATED AT 250 WORDS)

The initial phase of in vitro experiments in Ussing-type chambers on large intestine is characterized by short-circuit currents (ISC) declining from high starting values to a lower plateau within 0.5 h. The origin of this "initial ISC-transient" was investigated by ISC measurements on partially stripped segments of rat rectal colon. Transport was pre-stimulated in vivo by keeping animals in barbiturate-anesthesia for 5 h prior to tissue preparation. This procedure caused by endogenous aldosterone-liberation amiloride-sensitive Na-absorption to become the predominant electrogenic transport. The initial ISC-transient was abolished by tetrodotoxin (TTX, 1 microM), indicating a neuronal mediation of this phenomenon. In order to identify the transport which was subject to neuronal control, the amiloride-sensitive Na-absorption was measured during electrical field stimulation (bipolar rectangular pulses: 5 Hz, 1 ms, +/- 6 mA). There was no difference to unstimulated controls. In contrast, the initial ISC-transient was dependent on Cl in the bath following Michaelis-Menten-kinetics (KM = 20 mM) and could be prevented by 10 microM serosal bumetanide. Then, initial filling of the Ussing-chamber was imitated during the course of the experiment by removal and immediate re-addition of the bathing fluid. This procedure caused ISC-changes of similar appearance as the initial ISC-transient. To verify that indeed mechanical stretch is the sensory stimulus triggering the initial ISC-transient, the effect of small pressure oscillations was studied. This also produced an ISC-transient which was TTX-sensitive and was abolished after removal of the submucosal plexus Meissner by total stripping.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial and subepithelial resistance of rat jejunum was measured in vitro by two independent methods. (i) Transepithelial AC impedance data were interpreted in terms of a simple parallel RpCp element (representing the epithelial cell layer) in series with an ohmic resistor RS (representing the subepithelial layers). (ii) In separate experiments, the tip of a microelectrode was positioned between epithelium and subepithelial layers and the respective resistances were obtained from DC-pulse voltage divider ratios between both structures. The total tissue resistance as measured in conventional Ussing-chamber experiments (49 +/- 4 Ohm X cm2, mean of both methods) was formed to 81 +/- 6% (40 +/- 3 Ohm X cm2) by subepithelial layers and to only 19 +/- 3% (9 +/- 1 Ohm X cm2) by the epithelial cell line. We conclude that rat jejunum is more conductive than assumed so far. In in vitro flux studies on intact jejunal sheets a pronounced back-diffusion of absorbed substances will lead to an underestimation of the true net transport capacity of this structure. This error averages about fivefold and will be found likewise in conventional short-circuit measurements.