The structural requirements for binding to the glucose/sorbose-transport system in the human erythrocyte were explored by measuring the inhibition constants, K(i), for specifically substituted analogues of d-glucose when l-sorbose was the penetrating sugar. Derivatives in which a hydroxyl group in the d-gluco configuration was inverted, or replaced by a hydrogen atom, at C-1, C-2, C-3, C-4 or C-6 of the d-glucose molecule, all bound to the carrier, confirming that no single hydroxyl group is essential for binding to the carrier. The binding and transport of 1-deoxy-d-glucose confirmed that the sugars bind in the pyranose form. The relative inhibition constants of d-glucose and its deoxy, epimeric and fluorinated analogues are consistent with the combination of beta-d-glucopyranose with the carrier by hydrogen bonds at C-1, C-3, probably C-4, and possibly C-6 of the sugar. Both polar and non-polar substituents at C-6 enhance the affinity of d-glucose derivatives relative to d-xylose, and d-galactose derivatives relative to l-arabinose, and it is suggested that the carrier region around C-6 of the sugar may contain both hydrophobic and polar binding groups. The spatial requirements at C-1, C-2, C-3, C-4 and C-6 were explored by comparing the relative binding of d-glucose and its halogeno and O-alkyl substituents. The carrier protein closely approaches the sugar except at C-3 in the d-gluco configuration, C-4 and C-6. d-Glucal was a good inhibitor, showing that a strict chair form is not essential for binding. 3-O-(2',3'-Epoxypropyl)-d-glucose, a potential substrate-directed alkylating agent, bound to the carrier, but did not inactivate it.

6-O-methyl-, 6-O-propyl-, 6-O-pentyl- and 6-O-benzyl-D-galactose, and 6-O-methyl-, 6-O-propyl- and 6-O-pentyl-D-glucose inhibit the glucose-transport system of the human erythrocyte when added to the external medium. Penetration of 6-O-methyl-D-galactose is inhibited by D-glucose, suggesting that it is transported by the glucose-transport system, but the longer-chain 6-O-alkyl-D-galactoses penetrate by a slower D-glucose-insensitive route at rates proportional to their olive oil/water partition coefficients. 6-O-n-Propyl-D-glucose and 6-O-n-propyl-D-galactose do not significantly inhibit L-sorbose entry or D-glucose exit when present only on the inside of the cells whereas propyl-beta-D-glucopyranoside, which also penetrates the membrane slowly by a glucose-insensitive route, only inhibits L-sorbose entry or D-glucose exit when present inside the cells, and not when on the outside. The 6-O-alkyl-D-galactoses, like the other nontransported C-4 and C-6 derivatives, maltose and 4,6-O-ethylidene-D-glucose, protect against fluorodinitrobenzene inactivation, whereas propyl beta-D-glucopyranoside stimulates the inactivation. Of the transported sugars tested, those modified at C-1, C-2 and C-3 enhance fluorodinitrobenzene inactivation, where those modified at C-4 and C-6 do not, but are inert or protect against inactivation. An asymmetric mechanism is proposed with two conformational states in which the sugar binds to the transport system so that C-4 and C-6 are in contact with the solvent on the outside and C-1 is in contact with the solvent on the inside of the cell. It is suggested that fluorodinitrobenzene reacts with the form of the transport system that binds sugars at the inner side of the membrane. An Appendix describes the theoretical basis of the experimental methods used for the determination of kinetic constants for non-permeating inhibitors.

It is shown that the 7-dehydrocholesterol reductase-catalysed conversion of 7-dehydrocholesterol into cholesterol (II), with a 105000g microsomal pellet of rat liver in the presence of [4-(3)H(2)]NADPH, results in the transfer of radioactivity to the 7alpha-position of cholesterol. When the conversion is carried out in the presence of tritiated water the label is introduced exclusively at the 8beta-position. However, when the conversion of 7-dehydrocholesterol into cholesterol is performed with a 500g supernatant of rat liver homogenate the radioactivity is incorporated at both the 7alpha- and the 8beta-position. Evidence is provided for the presence of an enzyme system in the 500g supernatant that catalyses an equilibration of hydrogen atoms between those at the 4-position of NADPH and those of water. The work with stereospecifically labelled cofactors shows that both the equilibrating system and the 7-dehydrocholesterol reductase utilize the 4B-hydrogen atom of NADPH. In the light of these results a mechanism for the reduction of carbon-carbon double bonds is discussed.

Fluid transfer by isolated everted sacs of rat jejunum, ileum and intact colon prepared from adrenalectomized-nephrectomized rats 48 h after operation was reduced when compared with that of sacs prepared from untreated controls (P < 0\m=.\001).Angiotensin at 10\ m=-\ 10 g/ml significantly (P < 0\m=.\01) stimulated fluid transfer by intestinal sacs prepared from the adrenalectomized-nephrectomized rats; all three regions of gut were equally sensitive.Fluid transfer was similarly reduced in stripped colon sacs prepared from adrenalectomized-nephrectomized rats. Angiotensin had a dose-dependent biphasic action on fluid transfer by stripped colon sacs: low concentrations (10\m=-\11 and 10\m=-\12g/ml) stimulated (P < 0\m=.\05), whilst high concentrations (10\ m=-\ 9 and 10\m=-\8 g/ml) inhibited fluid transfer (P < 0\m=.\01).Histological examination of the colon preparations showed that the stripping procedure removed the ganglia, indicating that both angiotensin effects were due to direct action on the colon mucosa.The significance of these results is discussed in relation to the role of angiotensin in the control of salt and fluid transport by the mammalian kidney and other epithelial tissues.

SUMMARY1. A method has been described for the measurement of fluid transport by rat jejunum in vivo over two consecutive 30 min periods.2. Subpressor infusion rates of angiotensin (0.59 ng/kg per minute) stimulate fluid transport, while high (pressor) infusion rates (590 ng/kg per minute) inhibit fluid absorption.3. Both the inhibitory and stimulatory effects of angiotensin on fluid transport are not accompanied by any change in the transmural p.d., total blood flow to the jejunum or distribution of blood flow within the wall of the jejunum.4. These results are discussed in relation to the mechanism of action of angiotensin on fluid transport and its role in sodium and water homoeostasis.

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Epithelial sheets from rat jejunum and descending colon have been shown to respond to angiotensin II (AII) when studied under short‐circuit conditions and bathed on both sides with Krebs‐Henseleit solution. The octapeptide AII elicited increases in short‐circuit current (SCC) in preparations of jejunum and decreases in SCC in the descending colon; both responses occurred when the peptide was applied to the basolateral surface, but not when applied to the apical solution. Responses in both tissues were highly specific, being inhibited by a range of AII antagonists with the following order of potency: [Sar1. Thr8]‐AIi>[Sar1. Leu8]‐AIi>[Sar1. Ile8]‐AIi>[Sar1. Ala8]‐AIi>[Des, Asp1. Ile8]‐AII in rat jejunum. AII responses were not affected by α‐or β‐adrenoceptor antagonists, atropine or tetrodotoxin. AII responses were totally inhibited by the chloride channel blocker, diphenylamine‐2‐carboxylate (DPC) while cotransport inhibitors e.g. piretanide and frusemide significantly reduced the size of AII responses in colon and jejunum. These patterns of activity suggest that in the jejunum the responses result from electrogenic chloride secretion. Although AII responses in colon were sensitive to DPC the transporting ions have not yet been identified. Both piroxicam and indomethacin inhibited the increase in SCC elicited by AII in the jejunum, and the reduction in SCC caused by AII in the colon. Taken together these results indicate that eicosanoids are involved in AII responses in both tissues. This is the first study to demonstrate a direct, electrogenic effect for AII on transporting epithelia from the gastrointestinal tract. The responses are most probably initiated by AII interacting with previously identified specific AII receptors within the epithelial membranes.