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We have recently reported the results of a 24-month, double-blind, placebo-controlled study in 359 elderly osteoporotic women who were treated with daily oral alendronate (ALN) 1, 2.5, or 5 mg or placebo (PBO). We report the results of a 12-month, open-label, extension study during which 246 patients from the original study were treated with ALN 10 mg/day. Significant increases in lumbar spine bone mineral density (BMD) were observed in patients who had previously received PBO or ALN 1 and 2.5 mg/day for 24 months. Significant gains in trochanter BMD were seen in all treatment groups. Small changes were observed in femoral neck, total body, and forearm BMD during the course of this extension study. In general, the greatest increases in BMD during the open-label extension year occurred in patients who received either PBO or the lower doses of ALN during the previous 2-year blinded study. The frequencies of all categories of upper gastrointestinal adverse experiences (AEs) were less during months 25–36 (open-label extension) than during months 0–24 (original study). In conclusion, treatment with ALN 10 mg/day for 12 months in elderly women with osteoporosis who were previously treated for 24 months with PBO or ALN 1, 2.5, or 5 mg/day increased or maintained BMD of the spine, trochanter, and forearm, and was generally safe and well tolerated, especially in the upper gastrointestinal tract.

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.

1. The subcellular distribution of cyclic AMP in guinea-pig cerebral-cortical tissue was determined before and after electrical stimulation, and after a period of continued incubation after electrical stimulation, with and without the presence of histamine. 2. Electrical stimulation and histamine increased the cyclic AMP content of all fractions, the greatest increase occurring in the supernatant fraction. 3. Continued incubation after cessation of electrical stimulation diminished the cyclic AMP of the total homogenate and supernatant fractions, but increased that of the synaptosomal fraction. 4. Further fractionation of the synaptosomal fractions from stimulated tissues suggested that most of their soluble cyclic AMP was in a higher-molecular-weight form than was that of the tissue supernatant. 5. It is suggested that protein binding and cytoplasmic transport of cyclic AMP are involved in the changes observed.

1. The Na(+)-plus-K(+)-stimulated adenosine triphosphatase [(Na(+),K(+))-ATPase] of microsomal preparations from ox brain was inactivated or diminished in activity by exposure to 2-8m-urea. Similar concentrations of urea diminished the turbidity of the suspensions. 2. Low concentrations (about 2.5mm) of NaATP with the urea gave partial or complete protection of the ATPase, without altering the concomitant change in turbidity. Some protection of the (Na(+),K(+))-ATPase was afforded by tris ATP, but the greatest protection was found with NaATP and in its presence the change in (Na(+),K(+))-ATPase with 3m-urea included a phase in which activity was enhanced by 40%. 3. The protective effect was specific to NaATP: KATP, NaADP, NaAMP and sodium pyrophosphate were without protective effect and in some cases they augmented the action of urea. 4. The turbidity of cerebral microsomal suspensions was diminished also by ultrasonic irradiation; NaATP did not alter this change. After ultrasonic treatment up to 55% of the protein and of the ATPase activity were no longer deposited by centrifugal forces of 4.5x10(6)g-min. 5. Ultrasonic treatment and centrifugation could be carried out with little or no loss of ATPase and ammonium sulphate flocculation of the supernatant then afforded in the first material precipitated a three- to five-fold enrichment of (Na(+),K(+))-ATPase activity. 6. Sodium borohydride and dimethyl sulphoxide also diminished the turbidity of the microsomal fraction but enrichment of the ATPase was not effected by these reagents; ten other compounds were without action on the ATPase. 7. Acetyl phosphate was hydrolysed by the microsomal preparation and this activity was increased by added K(+). Acetyl-phosphatase activity persisted in the ultrasonically treated and ammonium sulphate-fractionated preparations, which were more exacting in their requirements for K(+). 8. The findings are discussed in relation to the mechanism of the (Na(+),K(+))-ATPase.

MEANS of electrically stimulating the metabolism of separated cerebral tissues have recently been developed in these laboratories.* The methods render such tissue much more susceptible to a wide variety of agents than are unstimulated tissues.3 and metabolic lesions, otherwise unapparent, may thus be detected. The methods are applicable to a few milligrams of tissue taken as biopsy specimens or during neurosurgical operations. They have not yet found a routine use, but their value in research is indicated by the following account, which describes briefly some results previously reported and adds others hitherto unpublished.A separated fragment of the mammalian cerebral cortex is electrically inert when examined by techniques which reveal profuse and intricate electrical activity in the brain in situ. A few seconds only after such a fragment has been removed from the brain, not only will it give no electrical response to applied electrical pulses, but also the

1. Uptake of [14C]adenine and [14C]adenosine from surrounding fluids to guinea-pig cerebral tissues was measured during incubation in vitro. Output of 14C-labelled compounds from the loaded tissues to superfusion fluids occurred on continued incubation, at about 0.2% of the tissue's content/min, and this rate was increased about fourfold by electrical excitation of the tissue. 2. The compounds released from the tissue to superfusion fluids included adenine, adenosine, inosine and hypoxanthine with small amounts of nucleotides. Output of all these compounds, except adenine, increased on excitation. Media depleted of oxygen or glucose also increased the output of 14C-labelled derivatives from [14C]adenine-loaded tissues, and this augmented output was further increased by electrical stimulation. 3. [14C]Adenosine was found as the main product from [14C]ATP when this was added at low concentrations to fluids superfusing cerebral tissue. Metabolic and neurohumoural explanations of the liberation and action of adenosine derivatives in the tissue are discussed.

1. Sample from the neocortex and piriform cortex of guinea pigs and rats were incubated in inulin-containing glucose–saline. Their intracellular (non-inulin) space contained 19–27μequiv. of Na+/g. of original tissue. These values were stable between 30 and 100min. after incubation commenced, but addition of 22NaCl to the neocortical samples showed them to be associated with a flux of 400μequiv. of Na+/g. of tissue/hr. 2. Addition of 0·5–10mm-l-glutamic acid or 0·1mm-N-methyl-dl-aspartic acid rapidly increased the tissue's Na+ content; N-acetyl-dl-aspartic acid was without action. 3. During the first 1–1·5min. after the addition of l-glutamic acid to neocortical samples their Na+ content increased at 600μequiv./g. of tissue/hr., and the rate of 22Na+ influx corresponded to 1230μequiv. of Na+/g./hr. These rates were calculated to be sufficiently rapid to account for loss of the tissue's normal membrane potential within 1–2sec. of the addition of the acid. 4. In addition, a rapid but more limited loss of K+ took place after the addition of l-glutamic acid or the methylaspartic acid; on continued incubation tissue K+ content increased, as also did the intracellular volume of the tissue, from its original 670μl./g. to 1100μl./g. 5. Interpretation of these and of associated changes is offered in terms that involve a cation pump and the permeability changes associated with the nerve impulse.

Small samples from the mammalian brain remain excitable in vitro, and spike discharges similar to many observed in vivoare evoked in neocortical samples. The autonomy of such samples will make them very valuable in investigations of the working of the brain.

1. Adenosine triphosphatase activities of dispersions prepared from bovine cerebral cortex that had been frozen, were greater than those of dispersions prepared from fresh tissue. The subcellular distribution of components of the dispersion was not altered by freezing the tissue and a microsomal fraction enriched in Na++K+-stimulated adenosine triphosphatase activity was prepared. 2. The bovine cerebral microsomes were further treated with a 2m-sodium iodide reagent to obtain a particulate preparation with minimal Na++K+-independent adenosine triphosphatase activity. Na++K+-stimulated activity was increased by the sodium iodide treatment and this preparation was shown to be enriched in lipid constituents. 3. Density-gradient centrifugation of the sodium iodide treated preparation gave three main subfractions each containing approximately equal amounts of phospholipid and protein. Further exposure of the sodium iodide-treated preparation to the 2m-sodium iodide reagent altered the distribution of protein and phospholipid among the fractions obtained by density-gradient centrifugation. Dissociation of phospholipids from protein in the sodium iodide-treated preparation was brought about also by high concentrations of arginine. Concentrated solutions of arginine and sodium thiocyanate brought about dissociation of phospholipids from protein of the microsomal preparation. 4. Many amino acids were found to inhibit Na++K+-stimulated adenosine triphosphatase activity when present in high concentrations. The inhibition was complex but resulted, in part at least, from diminished affinity for ATP and Na+ in the presence of the amino acids. 5. A non-ionic detergent, Lubrol W, solubilized up to 40% of the enzyme activity of the sodium iodide-treated preparation together with 30% of the protein and phospholipid in the preparation. Protein was released from the sodium iodide-treated preparation by pancreatic elastase but Na++K+-stimulated adenosine triphosphatase activity of the residue was diminished. Ultrasonic treatment of the sodium iodide-treated preparation failed to release a significant proportion of Na++K+-stimulated adenosine triphosphatase activity into a form not deposited by ultracentrifugation.

1. Salts of l-glutamate added to cerebral tissues maintained in glucose–saline–bicarbonate solutions cause the Na+ content of the tissues to increase rapidly and K+ to be lost. Entry of 22Na+ also is accelerated by l-glutamate and this acceleration is inhibited by low concentrations of tetrodotoxin. 2. Tissue Na+ content and its rate of increase after the addition of l-glutamate are affected by the Ca2+ of incubation media. 3. Very rapid and extensive entry of Na+ to the tissue is caused by EDTA, and a moderate entry by citrate and ATP. Calculations of the concentration of free Ca2+ in media after these additions indicate that Na+ entry is sometimes associated with low Ca2+ concentration, but that other substances, especially l-glutamate, act without greatly diminishing Ca2+ concentration. 4. Experiments with 2,4-dinitrophenol and valinomycin are also reported and aspects of the Na+ entry formulated and discussed.