vided first estimates of the individual membrane potentials and resistances. The cecal ... transport capacity, distinctly different from the neighboring proximal colon, and suited to ...... the effect of amiloride. Pfluegers Arch. 371: 99-108, 1977. 12.
Ion transport and electrophysiology in rabbit cecum W. CLAUSS, B. HOFFMANN, H. SCHÄFER, AND H. HÖRNICKE Institut für Veterinärphysiologie, Freie Universität Berlin, D-1000 Berlin 33; and Institut für Zoophysiologie, Universität Hohenheim, D-7000 Stuttgart 70, Federal Republic of Germany CLAUSS, W., B. HOFFMANN, H. SCHÄFER, AND H. HöRNICKE. Iontransport and electrophysiology in rabbit cecum. Am.
J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G1090-G1099, 1989.-We investigated the basic bioelectrical properties and
the transport of Na+, Rb+, and Cl- in the isolated cecal epithelium of the rabbit. Intracellular microelectrode recordings provided first estimates of the individual membrane potentials and resistances. The cecal epithelium was found to be a moderately tight epithelium with a high transepithelial potential and a relative high paracellular resistance. It has, however, a low transepithelial resistance caused by a high rate of transcellular
ion transport and a high transcellular conductance. Under short-circuit conditions Na+ and Cl-are absorbed, whereas Rb'" is secreted. Na+ absorption seems tobe mediatedvia an electrogenic mechanism with low amiloride sensitivity and is par-
substitution and therefore is referred to as Rb+ flux). Our study reveals an enormous absorptive capacity for Na+, a large conductance for Cl-, and a significant secretion of Rb+. The results from our equivalent analysis show that the rabbit cecum has electrical properties distinct from other distal intestinal epithelia. Glossary V.
Rt Isc Jrn-+s
J .......m
tially linked to Cl- transport. Cl- absorption is not influenced
Jnet
by secretagogues, and Rb+ secretion occurs via a transcellular
Va
raute. A basolateral K+ conductance was also found. The results
show rabbit cecum as an epithelium with a high electrolyte transport capacity, distinctly different from the neighboring proximal colon, and suited to absorb the !arge quantities of electrolytes that are present in the luminalliquid. electrolyte absorption; electrolyte secretion; short-circuit current; electrical equivalent analysis
of rabbit large intestine has been mainly investigated in the distal colon (22). Some information is available for the segments of the proximal colon (1, 4, 5, 23, 25), and few data are published about rabbit cecum (4, 14, 15, 24). In monogastric herbivores like rabbits, the cecum is an important site of microbial fermentation and nutrient absorption. The maintenance of suitable fermentation conditions despite the large amounts of short-chain fatty acids (acetate, propionate, and butyrate) requires distinct mechanisms for transepithelial electrolyte transport. Results from in vivo studies (19) indicate such special transport properties. However, for a detailed evaluation of these mechanisms, experiments have to be carried out under standardized conditions (isolated cecal epithelia, standard Ringer solutions, short-circuit conditions). To the best of our knowledge, only few data about Na+ and Cltransport are available for such conditions (4, 14, 15, 24). W e therefore designed a study in which we attempted to elucidate the basic mechanisms and rates of ion transport in the cecal mucosa. The present study demonstrates the basic bioelectrical properties of isolated rabbit cecum under different conditions and the unidirectional fluxes ofthe ions Na+, Cl-, and K+ (the latter measured by Rb+ THE ELECTROLYTE TRANSPORT
Gl090
Vb1 a
Ea
EP Ra Rb1 RP
Transepithelial potential difference Transepithelial resistance Short-circuit current Mucosal-to-serosal ion flux Serosal-to-mucosal ion flux Net ion transport Apical membranepotential Basolateral membrane potential Membrane resistance ratio (a = Ra/Rbl) Apical electromotive force Paracellular electromotive force Apical membrane resistance Baselateral membrane resistance Resistance of the paracellular pathway
MATERIALS AND METHODS
Animals and preparation. Male New Zealand White rabbits, weighing 3-4 kg, fed with a commercial rabbit diet and water ad libitum were used. Prior to the experiments all animals were adapted for at least 10 days on a 12-h light:dark cycle with lights on at 6:00 A.M. The distinct circadian rhythm of soft and hard feces production (17), which affects cecal electrolyte composition (19) and electrolyte transport properties in different parts of the rabbit large intestine (3, 4), required an exact timing of the experiments. Therefore all preparations were carried out at 10:00 A.M. during the soft feces period. The animals were killed by an intravenous injection of pentobarbital sodium (Nembutal, Abbott), and the abdomen was opened by a midline incision. The corpus ceci with 10-15 folds and ~20 cm in length was removed and opened along the mesenteric border. Figure 1 shows a drawing of the anatomic details. The portion of the cecum that was taken for the experiments is indicated by the hatched area. The cecal contents were carefully washed out with Ringer solution at room temperature, then the underlying serosal muscle layers were stripped off similar to the "partial mucosal strip" technique of Frizzell et al. (9). Figure 2 shows a cross section of the
0193-1857/89 $1.50 Copyright© 1989 the American Physiological Society
ELECTROLYTE TRANSPORT IN RASBIT CECUM
cecal wall and its different layers under intact conditions and after the muscle layers were dissected off. The figure shows that the epithelium consists of a folded structure with a single layer of epithelial cells lining the mucosal surface. The infoldings reach far down into the submucosallayers. The main portion of the entire wall consists of the circular and longitudinal muscles, which could be dissected off. Only some connective tissue remained on the serosal side after dissection. We therefore consider our preparation as sufficient to measure the properties of epithelial transport of rabbit cecum. For a detailed information about the anatomy and histology of rabbit cecum, readers are referred to the work of Snipes (26, 27). Chambers and tissue mounting. Six tissues were vertically mounted in modified Ussing chambers with a volAppendix vermi formes -
- - prox . Colon 1- 1 Ileum
---
Corpus ceci -
FIG. 1. Anatomy of rabbit cecum. Shaded area represents the portion of the tissues that were taken for the experiments in the Ussing chambers. No regional differences in tissue properties were found in that area.
'~
""':. ·· .... ..: . . .. '
·.
-
A
·" •
...."..
-
ume of 15 ml on each side and a serosal surface area of 1.32 cm2 • The openings of the chambers were designed to minimize edge damage. A thin layer of silicon grease was used to seal the tissue to the chambers so that very little pressurewas necessary to hold the tissue. Wehave shown in a previous paper that under these conditions virtually no edge darnage can be detected (4) . The tissues were bathed symmetrically with the appropriate Ringer solution, maintained at 37°C, and aerated and circulated by a gas Iift with the appropriate mixture. For microelectrode experiments, the tissue was either mounted vertically in a chamber with an area of 2 cm2 , as described by Wills et al. (30), or horizontally in a chamber with an opening of 0.62 cm2 • Electrical measurements. Each chamber was connected to a computer-controlled voltage clamp (5). Series resistance (10- 15 n.cm 2 ) was determined before mounting and automatically corrected during the experiment. Vt was measured via Ringer-agar bridges and calomel halfcells (lngold 303) with reference to the serosal solution. l sc was considered positive when flowing from mucosal to serosal solution and was passed through Ringer-agar bridges connected to Ag/AgCl electrodes in 3 M KCI. All isotope experiments were carried out under short-circuit conditions. Rt was determined by bipolar current pulses of 100 J.tA/cm2 of 500-ms duration or by a brief currentclamping of the tissues to zero. All electrical parameters ( Vt, I.c, and Rt) were printed out in regular intervals and stored for further analysis on floppy disk. A detailed
.... -_...' ".
• '"-
r
,.
8
2. Cross section of the wall of rabbit cecum. A: portion where t he serosal muscle layers have been dissected off. 8: intact tissue with muscle layers. FIG.
G1091
G1092
ELECTROLYTE TRANSPORT IN RABBIT CECUM
description and drawing of the computer-controlled system is given by Clauss et al. (5). For microelectrode experiments, a similar computer-controlled voltageclamp system (Biomedical Instruments, Germering) was employed and interfaced to a stimulus isolation unit and via a 12-bit A-D system (AC Copy, Aachen) to an Apple li microcomputer. Microelectrode measurements. Conventional microelectrodes were pulled from borosilicate glass (Hilgenberg, Malsfeld) and filled with 0.5 mM KCl. The tip resistance was 80-120 Mrl in NaCl Ringer solution, and the tip potential was basolateral Na+-K+ pump. Unlike the rabbit distal colon,
=
