Biomedical subjects

W M Moran

Publications and source records attributed to W M Moran.

8 recordsLinked to original sources

PubMed1999

A simple, inexpensive method for teaching how membrane potentials are generated.

We have developed a simple laboratory exercise that uses an inexpensive dialysis membrane (molecular weight cutoff = 100) to illustrate the generation of membrane potentials (Vm) across plasma membranes of animal cells. A piece of membrane approximately 2.0 cm2 is mounted in an Ussing-like chamber. One chamber half is designated cytosol and the other half external. Chamber sidedness helps students relate their findings to those of real cells. As in real cells, outward directed K+ concentration gradients [high cytosolic K+ concentration ([K+]c) and low extracellular K+ concentration] generate cytosol electrically negative Vm with a slope of approximately -45 mV/decade change in [K+]c. The polarity of Vm reflects the outward flow of potassium ions because flow of the larger counterion, H2PO4-, is restricted to the pores in the membrane. A slope less than Nernstian (<59 mV/decade) suggests that the membrane is slightly permeable to H2PO4-. Importantly, this facilitates teaching the use of the Nernst equation to quantify the relationship between ion concentration ratios across membranes and magnitude of Vm. For example, students use their data and calculate a permeability ratio PK/PH2PO4 that corresponds to a slope of approximately 24% less than Nernstian. This calculation shows that Nernstian slopes are achieved only when permeability to the counterion is zero. Finally, students use the concept of membrane capacitance to calculate the number of ions that cross the membrane. They learn where these ions are located and why the bulk solutions conform to the principle of electroneutrality.

Animals↗

PubMed1998

Luminal L-alanine stimulates exocytosis at the K+-conductive apical membrane of Aplysia enterocytes.

In Aplysia intestine, stimulation of Na+ absorption with luminal alanine increases apical membrane K+ conductance (GK,a), which presumably regulates enterocyte volume during stimulated Na+ absorption. However, the mechanism responsible for the sustained increase in plasma membrane K+ conductance is not known for any nutrient-absorbing epithelium. In the present study, we have begun to test the hypothesis that the alanine-induced increase in GK,a in Aplysia enterocytes results from exocytic insertion of K+ channels into the apical membrane. We used the fluid-phase marker horseradish peroxidase to assess the effect of alanine on apical membrane exocytosis and conventional microelectrode techniques to assess the effect of alanine on fractional capacitance of the apical membrane (fCa). Luminal alanine significantly increased apical membrane exocytosis from 1.04 +/- 0.30 to 1.39 +/- 0.38 ng. min-1. cm-2. To measure fCa, we modeled the Aplysia enterocyte as a double resistance-capacitance (RC) electric circuit arranged in series. Several criteria were tested to confirm application of the model to the enterocytes, and all satisfied the model. When added to the luminal surface, alanine significantly increased fCa from 0.27 +/- 0. 02 to 0.33 +/- 0.04 (n = 10) after 4 min. There are two possible explanations for our findings: 1) the increase in exocytosis, which adds membrane to the apical plasma membrane, prevents plasma membrane fracture, and 2) the increase in exocytosis delivers K+ channels to the apical membrane by exocytic insertion. After the alanine-induced depolarization of apical membrane potential (Va), there is a strong correlation (r = 0.96) between repolarization of Va, which reflects the increase in GK,a, and increase in fCa. This correlation supports the exocytic insertion hypothesis for activation of GK,a.

Alanine↗

PubMed1995

Effect of L-alanine and ouabain on membrane conductances and apical membrane potential in Aplysia intestine.

The proximal intestine of Aplysia californica was employed to assess the effect of alanine absorption on apical membrane K+ conductance (GKa) and basolateral membrane conductance (Gb) and the role of the electrogenic Na(+)-K(+)-adenosinetriphosphatase (Na+ pump) in the repolarization of apical membrane electrical potential difference (Va) after alanine-induced depolarization. Addition of 50 mM L-alanine (isosmotic substitution for mannitol) to the apical superfusate depolarized Va, reduced the ratio of apical to basolateral membrane resistances (Ra/Rb), and stimulated short-circuit current (Isc). Following these initial events, Va repolarized, Ra/Rb increased, and there was a slight decline in Isc. Apical high-K+ artificial seawater revealed an alanine-induced increase in GKa. Washout of alanine from the apical solution increased Ra/Rb above the prealanine control value. Thus alanine absorption is accompanied by an increase in Gb. Basolateral 0.1 mM ouabain abolished alanine-stimulated Isc but had little effect on Va ( < 3 mV depolarization) either before or after exposure to alanine. The repolarization of Va was not affected in tissues superfused with 0.1 mM basolateral ouabain for approximately 3 min even though the alanine-stimulated increase in Isc was abolished. Therefore, the electrogenic Na+ pump contributes minimally to the repolarization of Va in sea hare intestine. The origin of the hyperpolarization of Va resides therefore, at least in part, in the increase in GKa, which restores the driving force for Na(+)-alanine cotransport and prevents K+ accumulation in the enterocytes.

Alanine↗

PubMed1993

Cl- secretion by cultured shark rectal gland cells. III. Ca2+ regulation of apical membrane Cl- conductance.

Calcium ionophores (ionomycin and A-23187) were employed to assess the effects of increased intracellular Ca2+ concentration ([Ca2+]i) on apical membrane Cl- conductance (GaCl) and rate of transepithelial Cl- secretion in cultured shark rectal gland (SRG) cells. Apical 2 microM ionomycin induced dramatic changes in cellular electrophysiological properties: the apical membrane electrical potential difference (V(a)) depolarized from -66 mV to -46 mV, the fractional resistance of the apical membrane (fRa) decreased from 0.88 to 0.23, and the transepithelial electrical potential difference (Vab) increased slightly from +1.2 mV to +1.4 mV. These effects result from increased GaCl because apical low-Cl- shark Ringer (SR) depolarized V(a) by 32 mV and increased fRa from 0.23 to 0.36. Ionomycin-stimulated Vab or short-circuit current (Isc) results largely from increased Cl- secretion because approximately 80% of the increase in Isc is Cl- dependent. Establishing the Ca2+ dependence of ionophore activation of GaCl was confounded because apical low-Ca2+ SR [Ca2+ concentration ([Ca2+]) 1 mM to 0.1 microM] alone activated this conductive pathway. To establish the Ca2+ dependence of ionophore action, we assessed the effect of ionomycin on Isc in low-Ca2+ SR ([Ca2+] = 0.1 microM) and in SR. In low-Ca2+ SR, apical ionomycin stimulated Isc by 14.0 microA/cm2. In SR (normal [Ca2+]), ionomycin increased Isc further by 27.0 microA/cm2. Superfusing the basolateral surface with 2 microM ionomycin for 8-16 min failed to activate GaCl. In every case, subsequent superfusion of the apical surface with ionophore for 1.5-2 min activated GaCl. Bilateral 4 microM indomethacin (45-min superfusion) failed to block the ionomycin-induced GaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Cl- secretion by cultured shark rectal gland cells. II. Effects of forskolin on cellular electrophysiology.

Employing microelectrode techniques we have assessed the cellular electrophysiological properties of shark rectal gland (SRG) cells in primary culture. In the absence of secretagogues a 10-fold reduction in the Cl- concentration of the apical superfusate shark Ringer solution had little effect on either apical membrane electrical potential difference (Va) or fractional resistance (fRa), indicating little, if any, apical membrane Cl- conductance. Superfusing the basolateral surface with high-K+ shark Ringer solution (K+ increased 10-fold) depolarized the basolateral membrane electrical potential difference (Vb) by 43 mV, indicating that this barrier is largely K+ conductive. In addition, basolateral Ba2+ (5 mM) depolarized Vb by 12 mV and reduced fRa from 0.92 to 0.58, results consistent with a K(+)-conductive basolateral membrane in unstimulated SRG cells. Basolateral forskolin (10(-6) M) depolarized Va by 25 mV and caused a dramatic reduction in fRa from 0.97 to approximately 0.10. Under these conditions, a 10-fold decrease in apical superfusate Cl- concentration depolarized Va by 37 mV, revealing an adenosine 3',5'-cyclic monophosphate-induced apical membrane Cl- conductance. The time course of the forskolin-induced changes in Va and Vb suggests that the basolateral membrane K+ conductance increased and maintained the driving force for apical Cl- exit, as in other Cl(-)-secreting epithelia. These electrophysiological properties compare favorably with those of the perfused SRG tubule and indicate that SRG primary cultures are a suitable model for Cl(-)-secreting epithelia.

Animals↗

PubMed1988

Sugar-stimulated ion absorption is not different in seahare and vertebrate intestine.

We have reexamined the notion that sugars stimulate ion absorption differently in invertebrate and vertebrate intestine. In the seahare intestine, mucosal sugar presumably increases the rate of transcellular Na+ and Cl- absorption, whereas only transcellular Na+ absorption is increased in the vertebrate small intestine. Our data indicate that the seahare intestine responds to mucosal D-galactose like the vertebrate small intestine: namely, the apical membrane electrical potential difference depolarizes, the ratio of the mucosal to serosal membrane resistances decreases, and the short-circuit current (Isc) increases. Because mucosal substitution of tetramethylammonium for Na+ abolished the increased Isc, this stimulation resulted from an increase in rheogenic Na+ absorption. Unidirectional transepithelial Cl- fluxes indicate that mucosal D-galactose had no effect on the net Cl- flux under short-circuit conditions. Further, ion substitution experiments indicate that the apical membrane is K+ conductive rather than Cl- conductive as previously reported. These electrophysiological as well as parallel histological findings indicate that studies previously reported on the seahare intestine were in fact conducted on the esophagus.

Animals↗

PubMed1986

Transcellular sodium transport and intracellular sodium activities in rabbit gallbladder.

This study was designed to explore the relation between the rate of transcellular active Na+ transport by rabbit gallbladder epithelium, JNa, and the intracellular Na+ activity, (Na)c; the latter was determined by use of highly selective Na+ microelectrodes. The underlying strategy was based on the well-established observation that JNa is stimulated by the presence of bicarbonate in the bathing solutions. Our results confirm previous observations that the addition of bicarbonate to the bathing solutions results in a twofold increase in JNa. In the absence of bicarbonate, (Na)c averaged 16 mM. Within 2-4 min after the addition of bicarbonate to both bathing solutions, (Na)c increased to an average value of 22 mM and then gradually declined and by 15 min did not differ significantly from the value observed in the absence of bicarbonate. Thus, a twofold increase in JNa is not associated with an increase in (Na)c. These results are in accord with earlier observations on Necturus urinary bladder and small intestine and contradict the notion that an increase in the rate of active Na+ extrusion from the cell across the basolateral membrane in response to an increase in the rate of Na+ entry across the apical membrane is necessarily the result of a sustained increase in (Na)c.

Animals↗

PubMed1985

Kinetics of the effect of amiloride on the permeability of the apical membrane of rabbit descending colon to sodium.

The effects of the addition of graded concentrations of amiloride, (A)m, to the mucosal bathing solution on the permeability of the apical membrane of rabbit descending colon to Na (PmNa) were determined when the Na activity in the mucosal bathing solution, (Na)m, was 18, 32 or 100 mM. PmNa was obtained from current-voltage relations determined on tissues bathed with a high-K serosal solution before and after the addition of a maximally inhibitory concentration of amiloride to the mucosal solution as described by Turnheim et al. (Turnheim, K., Thompson, S.M., Schultz, S.G. 1983. J. Membrane Biol. 76:299-309). The results indicate that: (1) As demonstrated previously (Turnheim et al., 1983), PmNa decreases with increasing (Na)m. (2) PmNa also decreases hyperbolically with increasing (A)m. Kinetic analyses of the effect of amiloride on PmNa are consistent with the conclusions that: (i) the stoichiometry between the interaction of amiloride with apical membrane receptors that results in a decrease in PmNa is one-for-one; (ii) there is no evidence for cooperativity between amiloride and these binding sites; (iii) the value of (A)m needed to halve PmNa at a fixed (Na)m is 0.6-1.0 microM; and, (iv) this value is independent of (Na)m over the fivefold range studied. These findings are consistent with the notion that the sites with which amiloride interacts to bring about closure of the channels through which Na crosses the apical membrane are kinetically distinct from the sites with which (Na)m interacts to bring about closure (i.e., "self-inhibition"). In short, the effects of (Na)m and (A)m on PmNa in this tissue appear to be independent and additive.

Amiloride↗