Electron probe X-ray microanalysis of rabbit ciliary epithelium.

Rabbit iris-ciliary bodies were preincubated in control and experimental Ringer's solutions before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. After preincubation in a baseline bicarbonate-free Cl- Ringer's solution, the ciliary epithelial intracellular Na+, K+ and Cl- concentrations were estimated to be 15 +/- 3, 162 +/- 14 and 46 +/- 5 mmol kg-1 intracellular water, respectively. The water and elemental Na, K, Cl and P contents were similar in the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells. As expected, inhibition of the Na,K-exchange pump by preincubation with ouabain markedly increased the intracellular Na content, and markedly reduced the intracellular K content, verifying the validity of the experimental analysis. The Cl- channels of the NPE cells likely play a critical role in determining the rate of aqueous humor formation. Therefore, we have examined the effects of altering Cl- transport on the intracellular composition in this initial microprobe study of the ciliary epithelium. As expected, exposure to bicarbonate increased the intracellular Cl and water contents. Replacement of external Cl- by NO3- was twice as effective as replacement by gluconate in leaching Cl- out of the intracellular compartment. An unexpected finding was that NO3- replacement of internal Cl- substantially increased the intracellular Na and decreased the intracellular K content, possibly by stabilizing the Na,K-pump in the E1P form and inhibiting enzyme activity.

Effects of voltage clamping on epithelial cell composition in toad urinary bladder studied with x-ray microanalysis.

Toad urinary bladder epithelial cells were incubated in Na Ringer's with the serosal surface of the epithelium clamped at either +50 mV, 0 mV (short-circuited) or -50 mV with respect to the mucosal surface. Following incubation, portions of tissue were coated with an external albumin standard and rapidly frozen. Cryosections were freeze-dried and cell composition determined by x-ray microanalysis. Cell water and ion contents were unaffected when tissues were short-circuited rather than clamped close to their open-circuit potential difference (+50 mV). Incubation with vasopressin at +50 mV, and under short-circuit conditions, caused Na uptake without cell swelling or gain in Cl. Clamping at -50 mV resulted in uptake of water and ions, with considerable variation from cell to cell. These variations in cell composition were exacerbated by vasopressin. The greater the increase in water content, the greater the rise in cell Cl. However, there was no consistent pattern to the associated changes in cation contents. Most cells gained some Na. In some cells, this gain was accompanied by an increase in K. In others, the gain of Na was predominant and cell K content actually fell. At -50 mV with ouabain, many of the cells also gained water. As was found in our earlier study with ouabain under short circuit conditions (Bowler et al., 1991), there was considerable variation in the extent of the Na gain and K loss; some cells were largely depleted of K while in others the K content remained relatively normal. These results indicate differences between granular cells in the availabilities in the plasma membranes of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control.

Observations on 'fade': a complication of the contractile response of smooth muscle to a large dose of an agonist.

1. A study has been made of the time course of contraction of guinea-pig isolated ileum when suddenly exposed to a high concentration of acetylcholine, carbachol or histamine. With a sufficiently large dose there was a ;fading' of the abrupt initial contraction. A sustained contraction followed, provided that the agonist was not washed out of the organ bath.2. The fade produced by giving large equipotent doses of acetylcholine and histamine simultaneously was substantially greater than that obtained by giving either agonist alone. It was comparable to that produced by a double dose of acetylcholine or histamine. This result does not support any explanation of fade based on receptor occupancy.3. The extent of fade and the level of the sustained contraction were strongly affected by the calcium concentration of the bath fluid: the higher the calcium concentration the less was the fade. It is suggested that fade occurs when there is such intense stimulation of receptors that excitation-contraction coupling becomes temporarily less efficient due to depletion of calcium from a store.