Improved bleeding profile and tolerability of tibolone versus transdermal E2/NETA treatment in postmenopausal women with female sexual dysfunction.

OBJECTIVES: To compare the incidence of vaginal spotting/bleeding events and breast pain between therapy with tibolone 2.5 mg and continuous combined transdermal estradiol (E(2))/norethisterone acetate (NETA) 50 microg/140 microg after 24 weeks of treatment. METHODS: A double-blind, double-dummy, randomized, controlled trial was performed and assessments were performed at baseline, week 12 and week 24. Bleeding/spotting events were recorded in a daily diary. Breast signs and symptoms were collected as adverse events. RESULTS: A total of 403 women (mean age 56 years) were randomized. Bleeding/spotting events during weeks 1-12 with tibolone and E(2)/NETA were experienced by 16% and 56% of women, respectively (p < 0.001). The corresponding percentages during weeks 13-24 were 12% and 51%, respectively (p < 0.001). E(2)/NETA was significantly more likely than tibolone to be associated with vaginal hemorrhage (11% vs. 0%; p < 0.001) and breast signs and symptoms (11% vs. 4%; p = 0.015). Early discontinuations resulting from adverse events were significantly more common in the E(2)/NETA group than in the tibolone group (20% vs. 12%), primarily related to withdrawal due to vaginal hemorrhage (8% vs. 0%). CONCLUSIONS: Tibolone has a significantly better tolerability profile than transdermal E(2)/NETA as measured by vaginal bleeding, breast pain and treatment continuation.

Effects of osteoclastic resorption on bone surface ion composition.

Osteoclasts are responsible for resorption of bone mineral. To determine how osteoclasts alter bone surface ion composition, neonatal mouse bone cells were isolated and cultured in the presence of parathyroid hormone (PTH) on bovine cortical bone. Surface ion composition of the resulting osteoclastic resorption pits was compared with that of unresorbed bone, utilizing a high-resolution scanning ion microprobe. Cortical bone cultured with cells in the presence of PTH had numerous resorption pits. The unresorbed area adjacent to the pits had a ratio of surface 23Na/40Ca of 18.7 + 1.6 (mean counts per second of detected secondary ions +95% confidence interval) and 39K/40Ca of 2.3 + 2.2. At the base of the pits, the ratio of 23Na/40Ca was 1.0 + 2.0 and 39K/40Ca was 0.1 + 1.0 (each different from area adjacent to the pit, P < 0.001). The ratio of 23Na/39K in the unresorbed area was not different from that at the base of the pit. Thus osteoclasts induce a decrease in the ratio of surface ion composition of both 23Na/40Ca and 39K/40Ca but not 23Na/39K in bovine cortical bone. The elevated ratios of 23Na/40Ca and 39K/40Ca on the surface, but not at the base of the pits, indicate adsorption of medium ions onto the mineral. Because osteoclasts foster the release of bone Ca, these results indicate that osteoclastic resorption causes a greater, and approximately equal, release of both 23Na and 39K compared with 40Ca from bone mineral. Osteoclasts appear to remove nonselectively the surface mineral that had been exposed to the medium, uncovering underlying mineral.

Increased sensitivity to 1,25(OH)2D3 in bone from genetic hypercalciuric rats.

As a model of human hypercalciuria, we have selectively inbred genetic hypercalciuric stone-forming (GHS) Sprague-Dawley rats whose mean urine calcium excretion is eight to nine times greater than that of controls. A large component of this excess urine calcium excretion is secondary to increased intestinal calcium absorption, which is not due to an elevation in serum 1,25(OH)2D3, but appears to result from an increased number of intestinal 1,25(OH)2D3 receptors (VDR). When GHS rats are fed a low-calcium diet, the hypercalciuria is only partially decreased and urine calcium excretion exceeds intake, suggesting that an additional mechanism contributing to the hypercalciuria is enhanced bone demineralization. To determine if GHS rat bones are more sensitive to exogenous 1,25(OH)2D3, we cultured calvariae from neonatal (2- to 3-day-old) GHS and control rats with or without 1,25(OH)2D3 or parathyroid hormone (PTH) for 48 h at 37 degrees C. There was significant stimulation of calcium efflux from GHS calvariae at 1 and 10 nM 1,25(OH)2D3, whereas control calvariae showed no significant response to 1,25(OH)2D3 at any concentration tested. In contrast, PTH induced similar bone resorption in control and GHS calvariae. Immunoblot analysis demonstrated a fourfold increase in the level of VDR in GHS calvariae compared with control calvariae, similar to the increased intestinal receptors described previously. There was no comparable change in VDR RNA levels as measured by slot blot analysis, suggesting the altered regulation of the VDR occurs posttranscriptionally. That both bone and intestine display an increased amount of VDR suggests that this may be a systemic disorder in the GHS rat and that enhanced bone resorption may be responsible, in part, for the hypercalciuria in the GHS rat.

Parathyroid hormone (1-34) and Nleu8,18Tyr34-parathyroid hormone, (3-34) amide increase diacylglycerol in neonatal mouse calvaria.

Previously, we and others have presented evidence that a calcium second messenger system is involved in the action of parathyroid hormone (PTH) on bone. In the present report, the effects of PTH(1-34) and PTH(3-34)amide treatment on diacylglycerol (DG) in neonatal mouse calvaria are described. PTH(1-34) produced a rapid (within 5 minutes) increase in calvarial incorporation of 3H-arachidonic acid into DG. The effect was maximal at 0.1 nMPTH(1-34), the lowest concentration tested. The 3-34 amide analogue of PTH increased DG to the same extent as PTH(1-34). The effect was maximal at 10 nM PTH(3-34)amide, the lowest concentration tested. These concentrations were lower than those required to elicit maximal effects on bone resorption. In contrast to effects on cyclic AMP, where the 3-34 amide inhibited the increase elicited by PTH, combined treatment of calvaria with PTH(1-34) and PTH(3-34)amide did not inhibit effects on resorption or diacylglycerol.

Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.

Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors.

Second messengers in thrombin-stimulated bone resorption.

Characterized human thrombins and two commercial bovine thrombin preparations were examined for their effects on bone resorption and on the cyclic AMP and phosphoinositide second messenger systems in bone. Human alpha- and gamma-thrombins, as well as both bovine thrombin preparations, stimulated bone resorption in vitro, whereas catalytically inactivated human diisopropylfluorophosphate (DIP)-alpha-thrombin did not significantly stimulate resorption. Human alpha-thrombin and a commercial bovine thrombin preparation increased cyclic AMP production in fetal rat limb bones, but another bovine commercial thrombin preparation and gamma-thrombin did not. Except for DIP-alpha-thrombin, all thrombins increased production of inositol phosphates in fetal rat limb bones at concentrations that stimulated resorption. In time course studies, bovine thrombin increased label in inositol trisphosphate at 30 s, with decreasing effects at later times. Inositol monophosphate increased progressively over 30 min. Our results are consistent with thrombin-stimulated bone resorption being mediated at least partially through the inositol phosphate pathway.

Does amrinone inhibition of stimulated bone resorption involve Na+-Ca++ exchange?

Amrinone, a new cardiotonic agent, inhibits stimulated bone resorption in vitro. Pretreatment of neonatal mouse calvaria or fetal rat limb bones in culture with 2 X 10(-4)M amrinone for 6 hr reduces the response to a stimulatory agent added after washing out amrinone. This irreversible inhibition by amrinone could be blocked if the stimulatory agent was present together with amrinone during the pretreatment. To determine whether the early interaction of amrinone with this system involved an effect on a Na+-Ca++ exchange that may be required for stimulated calcium release from bone, the pretreatment effect of amrinone was examined under conditions of altered medium calcium or sodium concentration. If the calcium concentration was lowered during the 6 hr pretreatment, the inhibitory effect of amrinone was partially overcome; conversely increasing the calcium concentration during the pretreatment potentiated the effect of a submaximal concentration of amrinone. Although these results were consistent with amrinone blocking the Na+-Ca++ exchange mechanism, the effects of altering the medium sodium concentration during the 6 hr pretreatment were inconclusive. Therefore sodium-independent effects of amrinone on calcium transport cannot be ruled out. Amrinone also inhibited macromolecular synthesis in bone but this effect does not account for its ability to inhibit stimulated resorption.