Preclinical experience with two selective progesterone receptor modulators on breast and endometrium.

Org 31710 and Org 33628 are two highly selective progesterone receptor modulators (PRMs) with respect to their anti-progestational and anti-glucocorticoid activity. The compounds have been studied both in vitro and in vivo. Org 33628 has approximately four times stronger anti-progestational activity in vitro than does Org 31710, and in rats it is about 15 times more potent in the pregnancy interruption test. Two main indications for the use of PRMs are breast cancer and fertility regulation. The effects of both Org 31710 and Org 33628 were tested in relevant models for these indications. The effects of the two compounds on breast tumor development were assessed and in rats using the DMBA model. Their potency in menses induction was tested in monkeys on a 4-day regimen in the luteal phase, and after a single dose at day 21 of the normal cycle, and under a continuous progestin treatment using desogestrel. The compounds were also tested alone in a continuous low-dose regimen. The effects on follicular development and ovulation were determined by measuring estradiol and progesterone levels. Cycle control was monitored by daily vaginal swabs. In the DMBA model, Org 31710 at oral doses of 0.8, 2.0, and 5.0 mg/kg showed a clear dose-related reduction in tumor load. With the two highest doses, an even lower tumor load was seen after a 3-week treatment period compared to the tumor load at the start of treatment. Org 33628 showed a similar efficacy as Org 31710 at a dose of 2.0 mg/kg. RU 486 after oral treatment was two times less potent in this model than Org 31710 and Org 33628. The efficacy of menses induction using the 4-day regimen is dependent on the time of administration relative to the progesterone peak in the luteal phase. The highest efficacy is achieved in the descending part of the peak, at which a 100% success rate is found with a dose of 1 mg/kg of either Org 31710 or Org 33628. In Cynomolgus monkeys, at a single dose of 15 mg/kg of Org 31710 or Org 33628 in the luteal phase, menses induction was achieved only in 60% of the treatment cycles. Surprisingly menses induction can be achieved with a single dose that is about a ten-times lower when the monkeys are treated continuously with desogestrel. Cycle control is better at low than at high doses of antiprogestin in combination with daily dosing of 4 microg/kg desogestrel. Despite the difference in receptor affinity, no difference between Org 31710 and Org 33628 was found in menses induction. In the continuous low-dose (1 mg/kg) regimen with the PRMs, follicular development occurs normally while ovulation is inhibited. Ovulation is resumed shortly after stopping treatment, and a normal menses occurs after the first progesterone peak. Both compounds may be interesting options for the prevention and treatment of breast cancer and for fertility control.

Inhibition of human erythrocyte Ca2+-ATPase by Zn2+.

Recent investigations suggest that Ca2(+)-ATPase from fish gills is very sensitive to Zn2+ (Hogstrand et al., 1996. Am. J. Physiol. 270, R1141-R1147). The effect of free Zn2+ ion on the human erythrocyte plasma membrane Ca2(+)-ATPase was investigated to explore the possible extension of this finding to humans. Membrane vesicles were prepared and the Ca2(+)-ATPase activity was measured as Ca2(+)-stimulated ATP hydrolysis and as ATP-dependent Ca2+ transport. The Zn2+ ion inhibited the erythrocyte Ca2(+)-ATPase by reducing Vmax and increasing the K0.5. While in the Ca2+ transport assay only the Vmax was affected at lower Zn2+ concentrations (50-100 pM), reduction of Vmax was always accompanied by an affinity decrease in the ATP hydrolysis assay. The Ca2(+)-ATPase was found to be inhibited by Zn2+ at extremely low concentrations. The IC10 and IC50 for Zn2+, at a Ca2+ concentration of 1.0 microM, were estimated at 4 and 80 pM, respectively. Although the Ca2(+)-ATPase might be more sensitive in vitro than in vivo conditions, the results suggest that physiological concentrations of Zn2+ may reduce the activity of the erythrocyte Ca2(+)-ATPase. Furthermore, disturbance of Ca homeostasis may be a mechanism causing Zn toxicity during exposure.

Na(+)-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus.

It is concluded that Ca2+ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na+/Ca(2+)-exchange mechanism that is driven by the (transmembrane) Na+ gradient established by Na+/K(+)-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na+/Ca(2+)-exchanger next to the Ca(2+)-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca2+ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na+ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca2+ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca2+ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adapted to FW. Branchial Na+/K(+)-ATPase activity was two times higher in SW adapted fish.

Mechanisms of zinc uptake in gills of freshwater rainbow trout: interplay with calcium transport.

The uptake mechanism of Zn2+ through the gill epithelium of freshwater rainbow trout was investigated both in intact animals and in isolated basolateral membranes. Involvement of the apical Ca2+ uptake sites in Zn2+ uptake was examined in vivo by pharmacological manipulation of the apical Ca2+ permeability. The apical entries of Ca2+ and Zn2+, but not Na2+ and Cl-, were inhibited by addition of La to the water. Addition of 1.0 microM La reduced the influxes of Ca2+ and Zn2+ to 22 +/- 3 and 53 +/- 7% (mean +/- SE) of the control value, respectively. Injection of CaCl2 also reduced the branchial influxes of Ca2+ and Zn2+. This treatment decreased the influx of Ca2- to 45 +/- 4% of the control level and the Zn2+ influx to 68 +/- 5%. These results strongly imply that Zn2+ passes across the apical membrane of the chloride cells of the gills via the same pathway as Ca2+. The presence of an active basolateral transporter for Zn2+ was investigated in vitro on isolated basolateral membranes. There was no ATP-dependent or Na2+(-)gradient driven transport of Zn2+ at physiological Zn2+ activities. The same system was used to study potential effects of Zn2+ on the basolateral Ca2+(-)adenosinetri-phosphatase. Zn2+ was found to be a potent blocker of this transporter, causing a mixed inhibitory effect on the ATP driven Ca2+ transport at a free Zn2+ activity of 100 pM.

Effects of Zn(2+) on Ca (2+) uptake by mitochondria and endoplasmic reticulum in permeabilized tilapia gill cells.

An intracellular ATP-dependent Ca(2+) pumping mechanism, distinct from mitochondrial Ca(2+) accumulation, was identified within tilapia gill cells. Cell suspensions treated with 0.003% saponin, which selectively permeabilizes the plasma membrane, were used to characterize the Ca(2+) sequentering mechanisms as endoplasmic reticulum and mitochondria and to determine the effect of Zn(2+) on their Ca(2+) storing activity. Of the Ca(2+) taken up by the endoplasmic reticulum, 80% was released by IP3 (10 µmol l(-1)). The Ca(2+) pump of the endoplasmic reticulum was 2.5 times less sensitive to Zn(2+) (IC50=0.05 nmol l(-1)) than was the mitochondrial uptake mechanism (IC50=0.20 nmol l(-1)). The results indicate that Ca(2+) is stored predominantly within the endoplasmic reticulum at 0.1 µmol l(-1) and that this storing capacity is seriously attenuated by namomolar concentrations Zn(2+).

N-terminal and C-terminal fragments of the hormone stanniocalcin show differential effects in eels.

The effects of an N-terminal, a C-terminal, and a mid-fragment of stanniocalcin, the primary hypocalcemic hormone in fish, on plasma total and free (ionic) calcium levels and whole animal calcium influx were tested in eels. Both the N- and the C-terminal fragments were hypocalcemic, causing 18 and 12% reduction in plasma calcium in stanniectomized eels, respectively. With both fragments the hypocalcemic action is transient. The hypocalcemia caused by the C-terminal fragment, although more rapid, is not as pronounced as the hypocalcemic action of the N-terminal fragment. Only the C-terminal fragment reduced calcium influx. The hypocalcemic activity of the C-terminal fragment then can be explained by its effect on calcium influx. The N-terminal fragment appears to function in a different manner. The mid-fragment has no effect on plasma calcium or calcium influx. The different parts of the hormone are concluded to have different effects.

A passive immunization technique against the teleost hypocalcemic hormone stanniocalcin provides evidence for the cholinergic control of stanniocalcin release and the conserved nature of the molecule.

An in vivo bioassay based on 45Ca uptake from the ambient medium was used to test the efficacy of serum from rabbits immunized against trout stanniocalcin to passively immunize trout, tilapia, American eel, and guppy against endogenous stanniocalcin. The passive immunization was effective in all species. The fact that this procedure worked under both homologous and heterologous conditions, and in fish from different taxonomic infradivisions, is consistent with the view that the stanniocalcins in the four species examined share common antigenic determinants. The trout stanniocalcin antiserum had no effect on whole body calcium uptake (inCa2+) in stanniectomized eels, indicating that the effect of the antiserum was dependent on the presence of functional Stannius corpuscles. The technique was then used to show that the inhibitory effects that calcium loading and the injection of the cholinoreceptor agonist carbachol have on inCa2+ probably involve a catecholamine-induced release of endogenous stanniocalcin from the Stannius corpuscles.

Calcium transport in gill plasma membranes of the crab Carcinus maenas: evidence for carriers driven by ATP and a Na+ gradient.

A procedure was developed for the preparation of inside-out vesicles from plasma membranes isolated from the branchial epithelium of the green shore crab Carcinus maenas (L.). Procedures normally applied to fish branchial epithelium required the introduction of an additional hypotonic shock to obtain a preparation containing 22% inside-out vesicles, 33% right-side-out vesicles and 45% leaky membrane fragments. In such membrane preparations, the first direct evidence for uphill (against a [Ca2+] gradient) ATP-dependent and Na(+)-gradient-dependent Ca2+ transport in crustacean gills was found. The affinity for Ca2+ of the ATP-driven Ca2+ transporter was 149 nmol l-1 and that of the Na+/Ca2+ exchanger was 1.78 mumol l-1; the Vmax values were 1.73 and 9.88 nmol min-1 mg-1 protein respectively. The relative importance of these carriers for Ca2+ transport in the branchial epithelium of the crab is evaluated on the basis of their calcium kinetics.

Kinetics of ATP- and Na(+)-gradient driven Ca2+ transport in basolateral membranes from gills of freshwater- and seawater-adapted tilapia.

Plasma membranes of the gills of freshwater- and seawater-adapted tilapia were analyzed for Ca(2+)-ATPase and Na+/Ca2+ exchange activity. The relative importance of ATP-driven and Na(+)-gradient-driven Ca2+ transport in Ca2+ extrusion was evaluated on the basis of kinetic analyses in vitro. The Na+/Ca2+ exchangers in branchial membranes from freshwater or seawater fish displayed similar kinetics. The ATP-driven Ca2+ pump, however, showed a somewhat lower affinity for Ca2+ in membranes isolated from seawater gills than in membranes from freshwater gills; no difference in Vmax was found. The activity of the exchanger was estimated to be 50% of that of the ATP-driven pump at prevailing cytosolic Ca2+ concentrations (10(-7) mol l-1). Opercular ionocyte densities and branchial Na+/K(+)-ATPase content were not significantly different in fish residing in fresh water or sea water. We conclude that the gills of tilapia living for prolonged periods in fresh water or sea water do not differ in the make-up of their basolateral membrane with regard to Ca(2+)-ATPase, Na+/Ca2+ exchange and Na+/K(+)-ATPase activity. Apparently, the densities of these carriers suffice for calcium and sodium homeostasis under these vastly different ambient conditions.

Studies on stanniocalcin: characterization of bioactive and antigenic domains of the hormone.

Stanniocalcin (STC) decreases branchial Ca(2+)-uptake in fish. In order to determine its bioactive domain, synthetic fragments (U amino acids (aa) 1-20; V aa 103-136; W aa 202-231) of eel STC were tested for their effect on Ca2+ uptake in tilapia (Oreochromis mossambicus). Ca2+ uptake was inhibited by an N-terminal fragment but not by a midfragment nor a C-terminal fragment of the mature hormone. We provide theoretical and experimental evidence that a midportion of STC, which is included in the synthetic fragment V, is the most antigenic site of the molecule. Polyclonal antibodies against stanniocalcin are directed against this midportion although this region of STC appears not to be essential for signal transduction. These results suggest that the currently available antibodies will recognize inactive STC fragments in the circulation. We conclude that the bioactive portion of STC does not correspond with the major antigenic portion of the hormone. The results imply that studies on plasma STC levels employing a polyclonal antiserum against STC should be interpreted with care.

Branchial calcium uptake: possible mechanisms of control by stanniocalcin.

The branchial Ca(2+) uptake by teleost fish is under inhibitory control by the hormone stanniocalcin (STC) which is generated by the corpuscles of Stannius (CS). Removal of the CS in North American eel, Anguilla rostrata LeSueur, induced a rapid rise in blood calcium levels. Branchial Ca(2+) influx following the extirpation of the CS (stanniectomy, STX) increased during the first four days and stayed elevated thereafter (in agreement with previous studies). The transepithelial potential (TEP) across the gills did not change after STX and this means that the electrochemical gradient for Ca(2+) is less favourable for passive influx of Ca(2+) in STX eel. Therefore, the Ca(2+) influx in STX eels is a transcellular flux, with Ca(2+) crossing the apical and basolateral membrane barrier. The kinetics of ATP-driven Ca(2+)-transport across basolateral plasma membranes from eel gills did not change after STX. Thus, the increased Ca(2+)-influx after STX is not correlated with changes in ATP-dependent Ca(2+)-extrusion across the basolateral membrane, suggesting a regulation at the apical membrane. Moreover, STC did not affect ATP-driven Ca(2+)-transport in isolated basolateral membranes (in vitro). STC (0.1 nM) reduced cAMP levels in dispersed eel gill cells. It had no significant effect on the IP3 levels in these cells. We postulate that STC controls the permeability to Ca(2+) of the apical membranes of the Ca(2+) transporting cells of fish gills by controlling second messenger operated Ca(2+) channels in the apical membrane.

Inhibition of Ca2+ uptake in freshwater carp, Cyprinus carpio, during short-term exposure to aluminum.

In carp exposed to pH 5.2 in fresh water, the Ca2+ influx from the water is reduced by 31% when compared to fish in water of neutral pH. At pH 5.2, the Ca2+ influx but not Na+ uptake is decreased by aluminum (Al). Al reduces Ca2+ influx dose-dependently: a maximum 55% reduction was observed after 1-2 h exposure to 200 micrograms.1(-1) (7.4 microM) Al. Branchial Ca2+ efflux is less sensitive to Al and affected only by exposure for more than 1 h to high Al concentrations. Na+ influx is not affected by concentrations Al up to 400 micrograms.1(-1). Na+ efflux, similarly to Ca2+ efflux, increased when fish were exposed for more than 1 h to 400 micrograms.1(-1) Al.

Cadmium inhibition of the erythrocyte Ca2+ pump. A molecular interpretation.

The effects of cadmium (Cd2+) on transmembrane Ca2+ transport and on the membrane permeability for Ca2+ were studied in human erythrocytes. The erythrocyte Ca2+ pump is inhibited competitively by Cd2+ via interaction with the Ca2+ transport site of the carrier and not via interaction with its activator calmodulin. The affinity of the Ca2+ pump for Cd2+ is extremely high (KI = 2.0 nM Cd2+). Cd2+ (less than or equal to 10(-4) M) does not alter the membrane permeability for Ca2+. We conclude that the pivotal mechanism in the toxic action of Cd2+ is the inhibition of Ca2+-ATPase mediated Ca2+ extrusion. As a result Cd2+ disturbs intracellular Ca2+ homeostasis and may increase cytosolic Ca2+ (Ca2+i) to toxic levels.

Cadmium inhibits plasma membrane calcium transport.

The interaction of Cd2+ with the plasma membrane Ca2+-transporting ATPase of fish gills was studied. ATP-driven Ca2+-transport in basolateral membrane (BLM) vesicles was inhibited by Cd2+ with an I50 value of 3.0 nM at 0.25 microM free Ca2+, using EGTA, HEEDTA and NTA to buffer Ca2+ and Cd2+ concentrations. The inhibition was competitive in nature since the K0.5 value for Ca2+ increased linearly with increasing Cd2+ concentrations while the Vmax remained unchanged. The Ca2+ pump appeared to be calmodulin dependent, but we conclude that the inhibition by Cd2+ occurs directly on the Ca2+-binding site of the Ca2+-transporting ATPase and not via the Ca2+-binding sites of calmodulin. It is suggested that Cd2+-induced inhibition of Ca2+-transporting enzymes is the primary effect in the Cd2+ toxicity towards cells followed by several secondary effects due to a disturbed cellular Ca2+ metabolism. Our data illustrate that apparent stimulatory effects of low concentrations of Cd2+ on Ca2+-dependent enzymes may derive from increased free-Ca2+ levels when Cd2+ supersedes Ca2+ on the ligands.

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout.

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.

Cadmium inhibition of Ca2+ uptake in rainbow trout gills.

The effects of cadmium (Cd2+) on calcium (Ca2+) transport in the gills of rainbow trout (Salmo gairdneri) were studied. The gill epithelium of freshwater fish represents a model for a Ca2+-transporting tight epithelium. Unidirectional Ca2+ fluxes in the gills were estimated in an isolated saline-perfused head preparation. Ca2+ influx was not affected when up to 10 microM Cd were added to the ventilatory water at the start of flux determinations (in vitro exposure). However, after 16 h in vivo preexposure of the fish to 0.1 microM Cd in the water, a 79% inhibition of Ca2+ influx was observed. Ca2+ efflux was not affected when up to 10 microM Cd were added to the ventilatory water during the flux determination. Ca2+ efflux in fish preexposed to 0.1 microM Cd for 16 h was also not affected; a preexposure to 1 microM Cd, however, resulted in a 173% increase in Ca2+ efflux rates. Tracer retention in the gill tissue indicated that both Ca2+ and Cd2+ enter the gill epithelium via a lanthanum (La3+)-inhibitable pathway. It is concluded that Cd2+ readily enters the branchial epithelial cells, similarly as Ca2+ does via La3+-sensitive apical Ca2+ channels. The inhibitory action of Cd2+ on transepithelial Ca2+ influx seems to result from an inhibition of the basolateral Ca2+ transport, occurring after a critical intracellular Cd2+ concentration has been reached.

Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium.

The interactions of Cd2+ with active Ca2+ transport systems in rat intestinal epithelial cells have been investigated. ATP-driven Ca2+ transport in basolateral plasma membrane vesicles was inhibited by Cd2+ with an I50 value of 1.6 nM free Cd2+ at 1 microM free Ca2+, using EGTA and HEEDTA to buffer Ca2+ and Cd2+ concentrations, respectively. The inhibition was competitive in nature since the Km value of Ca2+ increased with increasing Cd2+ concentrations while the Vmax remained constant. Cd2+ had similar effects on ATP-dependent Ca2+ uptake by permeabilized enterocytes, indicating that non-mitochondrial and mitochondrial Ca2+ stores are also inhibited by nanomolar concentrations of Cd2+. We conclude that ATP-driven Ca2+ transport systems are the most sensitive elements so far reported in Cd2+ intoxication.

Kinetics of ATP-dependent Ca2+ uptake by permeabilized rat enterocytes. Effects of inositol 1,4,5-trisphosphate.

Isolated rat enterocytes were permeabilized by saponin treatment. 45Ca2+ was accumulated by these cells when provided with ATP in a medium containing Ca2+ ligands. The use of oxalate, vanadate and mitochondrial inhibitors indicated that both non-mitochondrial and mitochondrial pools are involved. Kinetic analysis of non-mitochondrial Ca2+ uptake revealed a Km of 0.1 microM Ca2+ and a Vmax of 0.4 nmol Ca2+/mg protein X min for this Ca2+-pumping ATPase activity. Mitochondria started to take up Ca2+ between 0.2 and 0.3 microM free Ca2+ reaching maximal rates around 2 microM. At 1 microM free Ca2+ mitochondria accumulated 20 times more Ca2+ than the non-mitochondrial pool. Inositol 1,4,5-trisphosphate released 40% of the Ca2+ content of the non-mitochondrial pool. Half-maximal release was observed at 0.5 and 1.5 microM IP3 in duodenal and ileal cells respectively. These findings support the possibility that the phosphatidyl inositide metabolism plays a role in regulation of electrolyte transport in enterocytes.