A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na+,K+)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 2:1:1. The presence of sealed vesicles was also evident from the osmotic sensitivity of the D-[1-14C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization. Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 microns/sec at 35 degrees C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiac-glycoside digitoxin. These characteristics are consistent with the activity of a (Na+,K+) pump operating in inside-out vesicles.

The epithelial Na+ channel (ENaC) functions as the rate-limiting factor in aldosterone-regulated transcellular Na+ transport. In the study described here, the effect of aldosterone on ENaC mRNA levels, protein synthesis and benzamil-sensitive Na+ transport was investigated using primary cultures of immunodissected rabbit kidney connecting tubule and cortical collecting duct cells (CNT and CCD, respectively). After a lag time of 3 h, aldosterone caused transepithelial Na+ transport to increase, reaching maximal level of 260+/-44% after 16 h of incubation. The alpha, beta and gamma rabbit ENaC (rbENaC) mRNA levels, measured by semi-quantitative reverse transcriptase-polymerase chain reaction, were not changed by aldosterone during the first 3 h, but a twofold increase was apparent after 6 h; levels remained elevated for up to 16 h of incubation. Immunoprecipitation of [35S]methionine-labeled rbENaC revealed a rise in protein levels of the alpha and beta subunits, but the protein level of the gamma subunit remained constant. In conclusion, our data suggest that in rabbit CNT and CCD the early increase in Na+ transport caused by aldosterone is due to the activation or insertion of existing Na+ channels into the apical membrane, and that the late response is mediated by increased synthesis of the alpha and beta rbENaC subunits.

Aquaporins are transmembrane proteins that contain six bilayer-spanning domains, connected by loops A to E. The hourglass model predicts that the conserved loops B and E are essential for the formation of the water pore. To test the importance of loops B and E in the determination of the transport characteristics, we exchanged loops B and/or E between AQP0, AQP2, and AQP3. Detailed functional, immunoblot and immunocytochemical analyses of expression in Xenopus oocytes revealed that six out of the nine chimeric aquaporin proteins were not functional, because of misrouting. AQP0 with loop E of AQP2 was not impaired in its routing and revealed a low water permeability equal to that of wild-type AQP0. AQP2 with loop B of AQP0 was also routed normally and gave a high water permeability, similar to that of wild-type AQP2. AQP0 with loops B and E of AQP2 (AQP0–2BE) did not result in an increase in water permeability and was partly misrouted. However, the plasma membrane expression was high enough to expect an increase in water permeability, as loops B and E of AQP2 confer AQP2’s water permeability to AQP0. Although it is unclear for the dual chimera (AQP0–2BE), the parental water permeabilities obtained in oocytes expressing AQP0 with loop E of AQP2 or AQP2 with loop B of AQP0 indicate that, besides loops B and E, other parts of the AQP protein are important in the determination of the characteristics of the channel.

Effects of biogenic amines on silage intake and rumen fermentation during dietary changes were studied in sheep. Two direct-cut grass silages were prepared from a single grass sward, one untreated (WAS) and one treated with 4.0 litres formic acid (850 d) per tonne (FAS). Diets of FAS, and FAS supplemented with 7.2 g biogenic aminedkg DM (FAS +A), were offered ad libitum, once daily to four rumen-cannulated, and four intact wethers in a repeated crossover design experiment. During a pre-period before each crossover, the animals were offered either the silage low in biogenic amines (FAS), or that containing moderate concentrations (WAS). During the first 4 d of the FAS + A treatment, the added biogenic amins tended to lower daily DM intake (DMI) and lowered significantly the DMI during the principal meal after feeding. This acute effect on DMI tended to be reduced when the sheep were previously preconditioned to amines by feeding WAS, and the acute DMI depression during the principal meal was significantly reduced. At the end of the 14 d FAS + A feeding period daily DMI was similar to that of the FAS treatment, but the daily pattern of intake remained Merent, with lower intake of FAS + A during the first 5 h after feeding, this being compensated for by the end of the day. Rumen fermentation tended to be less during the first 4 d that FAS +A was offered, due to the lower DMI and not due to the acute effect of amines.However, in the sheep unadapted to FAS + A, amine content in the rumen was higher than when the sheep were adapted for 14 d to FAS + A or WAS. Adaptation to FAS + A and feeding WAS during the pre-periods, increased the amine-degrading capacity of rumen fluid. In conclusion, in sheep unadapted to dietary amines, feeding amines will acutely lower DMI through reduced palatibility and most probably by stressing intermediary metabolism. Being preconditioned to amines slightly reduces the acute effect on daily DMI. Although the sheep adapted within 14 d to biogenic amines in the diet and increased daily DMI, there was clear evidence that amines have a negative effect on palatability.Biogenic amines: Fixed intake: Grass silage: Sheep Biogenic amines are thought to be one of the fermentation products which lower intake of ensiled forages, either by reducing palatability or by causing stress in intermediary metabolism after being absorbed (Beever & Reynolds, 1994). However, studies with sheep (Van 0 s et a2. 1995a, 1996a) and dairy cows (Van 0 s et al. 199%) showed that concentrations of amines (7 g/kg DM), comparable to those present in poor-quality grass silages, did not affect intake. In those studies, animals were adapted to the dietary treatment for 14 d before intake behaviour was monitored. However, some animals did show reduced silage intake during the first days after the change-over from a silage low in biogenic amine content to the same silage supplemented with biogenic amines. Possibly biogenic amines

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