Localization of different alcian blue-proteoglycan particles in the intervertebral disc

A histochemical investigation was carried out on proteoglycans of bovine intervertebral disc. Samples obtained from the "annulus fibrosus" (A.F.) and "nucleus pulposus" (N.P.) were treated with Alcian blue (AB) diluted in solutions of MgCl2 at critical electrolyte concentrations (CEC); some samples were incubated in testicular hyaluronidase before AB treatment. At least four types of elongated AB-proteoglycan particles were recognized: a) in A.F. lamellae and N.P., 1 nm rod-like particles were arranged orthogonally to the collagen fibrils and spaced at a distance equivalent to the fibril D-period (Figs. 1-5); b) within the A.F. lamellae, other 16-20 nm particles formed a close network among the collagen fibrils (Figs. 1,2,3,5); c) in the A.F. interlamellar crevices, 30-50 nm leaf-like particles were present (Fig. 6); d) in the N.P.Z., 20-30 nm leaf-like particles formed a wide-mesh (Fig. 4). The alcianophylic particle sizes suggest they may correspond to proteoglycan monomers in the A.F. lamellae and mostly proteoglycan aggregates in A.F. interlamellar crevices and N.P.. Both alcianophylia degrees at MgCl2 CEC solutions and enzymatic susceptibility indicate the presence of chondroitin sulphate and keratan sulphate and that the large particles in the A.F. interlamellar crevices are the keratan sulphate richest proteoglycans. The features of the observed AB-proteoglycan particles are consistent with previous morphological data reported for other tissues as well as some biochemical data for the intervertebral disc and may be correlated to the composite mechanical properties of this tissue.     

Effects of experimental diabetes on spontaneous contractions, on the output of prostaglandins and on the metabolism of labelled arachidonic acid, in uteri isolated from ovariectomized rats. Influences of estradiol

Spontaneous changes in isometric developed tension (IDT) as a function of time after isolation (contractile constancy) in uteri from control-castrated and castrated chronic streptozotocin-diabetic rats, were explored. The effects of injecting 17-beta estradiol (Eo) were also studied. No differences in the minor changes of contractile constancy, between control and diabetic preparations, during a period of 60 min, were detected, whereas uteri from non-diabetic Eo injected animals (0.5 + 1.0 ug, prior to sacrifice), exhibited a profound reduction of IDT, significantly greater than in tissues obtained from Eo injected-diabetic rats. Moreover, basal generation and outputs into the suspending solution of prostaglandins (PGs) E1, E2 and F2 alpha, were explored in the same groups, at 60 min following tissue isolation. The basal outputs of these three PGs were similar in castrated control rats, but preparations from castrated-diabetics released significantly more PGE1. The administration of Eo to castrated-diabetics, failed to alter the releases of the three PGs explored. In addition, the metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF1, PGF2, PGE2 and thromboxane B2-TXB2), was also investigated. The non-diabetic spayed rat uterus converted AA into these four prostanoids, the transformation into 6-keto-PGF1 alpha (as an index of PGI2 formation) being the most prominent. In preparations from diabetic rats the formation) being the most prominent. In preparations from diabetic rats the formation of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, was significantly smaller than in controls, whereas a greater % of TXB2 formation (as an index of TXA2), was detected. On the other hand uterine preparations from non-diabetic spayed rats injected with Eo formed less 6-keto-PGF1 alpha and PGE2 and similar amounts of PGF2 alpha or of TXB2 from AA, than Eo injected controls, whereas uteri from castrated diabetic animals injected with Eo, formed a similar % of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from AA, than tissue preparations from non-estrogenized controls. However, the enhanced transformation of the labelled fatty acid precursor (AA) into TXB2 in the diabetic group, was significantly reduced by the steroid. The role of the augmented generation and release of PGE1 in uteri from diabetic rats is discussed in terms of precedents indicating the relevance of PGs type E supporting rat uterine motility. In addition the influence of Eo is attractive, because its reducing effect on TX production, in diabetes, a disease known to be accompanied by enhanced synthesis of vasoconstrictor and platelet aggregation TXA2, and by frequent obstructive circulat     

Histamine alters prostaglandin output from diestrous rat uteri. Involvement of H2-receptors and 9-keto-reductase

The effects of exogenous histamine (H) on prostaglandin (PG) generation and release in uteri isolated from diestrous rats and the influences of H2-receptors blockers (cimetidine and metiamide) on the output of uterine PGs, were explored. Moreover, the action of H on the uterine 9-keto-reductase, was also studied. Histamine (10(-4) M) failed to alter the basal output of PGE1 but reduced significantly the generation and release of PGE2 and augmented the output of PGF2 alpha. On the other hand, cimetidine (10(-5) M) enhanced the basal release of PGE2 but had no action on the outputs of PGs E1 or F2 alpha. The enhancing effect of H on the production and release of PGF2 alpha was abolished in the presence of cimetidine. Also, the antagonist reversed the influence of H on the output of PGE2. Metiamide, another H2-receptor antagonist, did not alter the basal control generation and release of uterine PGs, but antagonized the augmenting influence of H on PGF2 alpha uterine output, as much as cimetidine did, and prevented the depressive action of H on the release of PGE2 from uteri. Histamine (10(-4) M) significantly stimulated uterine formation of cyclic-adenosine monophosphate, an action which was antagonized by the presence of cimetidine (10(-5) M), a blocker of H2 receptors. Also, histamine (10(-5) M) and dibutyrylcyclic-adenosine monophosphate (DB-cAMP) at 10(-3) M, enhanced significantly the formation 3H-PGF2 alpha from 3H-PGE2. Results presented herein demonstrate that H is able to diminish the generation of PGE2 in uteri from rats at diestrus augmenting the synthesis of PGF2 alpha, apparently via the activation of H2-receptors, enhancing adenylate-cyclase. These effects appear to increase uterine 9-keto-reductase activity which transforms PGE2 into PGF2 alpha. Relationships between the foregoing results and those evoked by estradiol, are also discussed.     

Effects of morphine on arachidonic acid metabolism, on Ca2(+)-uptake and on cAMP synthesis in uterine strips from spayed rats

The effects of morphine on arachidonic acid metabolism, on cAMP levels and on basal and induced 45Ca2(+)-uptake, in uterine strips isolated from ovariectomized rats as well as the influence of naloxone, were explored. The presence of morphine (10(-6) M) did not change significantly 14C-arachidonic acid metabolism, basal cAMP levels, or cAMP increment induced by PGE2 or by PGE1. On the other hand morphine (10(-6) M) decreased basal uterine 45Ca2(+)-uptake as much as verapamil (10(-6) M) did, and this action was not prevented by naloxone (10(-8) M). The presence of oxytocin (50 mU.ml-1) augmented 45Ca2(+)-uptake, an effect which was antagonized by morphine (10(-6) M). This inhibitory action of morphine on oxytocin-induced 45Ca2(+)-uptake was not prevented by naloxone (10(-8) M). Furthermore, PGE1 (10(-8) M and (10(-6) M) but not PGE2 (10(-8) and 10(-6) M), stimulated the incorporation of 45Ca2+ into uterine strips, and this action was not altered by morphine. The inhibitory influence of morphine on uterine spontaneous motility and on prostaglandin synthesis and release, previously described by us, is now explained in terms of an inhibition of tissue Ca2(+)-uptake.     

Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series

We have previously characterized in Chinese hamster lung fibroblasts a growth factor activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of growth factor-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity 20- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon growth factor action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for growth factor-induced DNA synthesis.     

Sow (Sus scrofa) follicular fluid: prostaglandin content and effect on the motility of isolated oviducts

The present study provides information regarding the effects of the sow follicular fluid (FF) on the motility of isolated segments of swine and rabbit oviducts. In addition, the concentration of prostaglandins (PGs) F2 alpha, E2 and E1 in the follicular fluid of sow ovaries isolated at different stages of the sex cycle as well as the generation of the same PGs by walls of ovarian follicles in early and late proestrus, in estrus, in metestrus and in diestrus, were explored. The stimulatory contractile effect of proestrous FF in isolated segments of sow fimbria was antagonized by polyphloretin phosphate (PPP), a PG receptor blocker and by indomethacin, an inhibitor of PG synthesis. The positive inotropism evoked by the FF was mimiked by bradykinin and the influences of both interventions were similarly antagonized by PPP. It appears plausible that the inotropic effect of the preovulatory FF on the sow fimbria could be not only by PGs already present in the fluid, but also by the stimulation of the synthesis of tubal PGs by follicular fluid bradykinin. The FF also stimulated the ampullary tubal segments isolated from proestrous sows whereas the same volume of FF depressed significantly the isometric developed tension of rabbit ampulla. The total concentration of the three PGs in the FF from late proestrous follicles was significantly greater than that of the same PGs in the other two stages of the sex cycle (early proestrus and diestrus), whereas the concentration of each PG (PGE2, PGF2 alpha or PGE1), did not differ within any of the stages of the cycle. Furthermore, the total amount of the three PGs produced by the walls of follicles from late proestrous ovaries was also significantly greater than that generated by ovarian follicles from early proestrus, estrus, metestrus and diestrus. In summary the results document that the concentration of each one of the PGs measured (E2, E1 or F2 alpha) attained maximal values at the time of ovulation. The results regarding the effects of FF on the inotropic activity of fimbrial and ampullary segments of sow oviducts also suggest that the fluid might play a physiological role, favouring the capture and transfer of ova into the oviducts at the moment of ovulation.     

Long-term intramuscular administration of thyrotropin-releasing hormone tartrate in patients with cerebrovascular disease: effects on the pituitary-thyroid axis.

We studied the effects of long-term (30 days) refracted daily intramuscular administration of 4 mg TRH tartrate (TRH-T) on the pituitary-thyroid axis in 20 euthyroid patients affected by cerebrovascular disease (CVD). All subjects were assayed for T4, T3, FT4, FT3, TSH and TBG plasma levels before treatment (D0), after 15 and 30 treatment days (D15, D30), and after a 15-day washout (D45). In addition, TSH response to 200 micrograms intravenous TRH was assessed at D0, D30 and D45. We observed a significant increase in T4, FT4 and FT3 levels in the face of decreased TSH concentrations. A blunted TSH response to TRH bolus persisted at D30. These data demonstrate that the down-regulation mechanism may be partially overcome in vivo when thyrotrophs are chronically exposed to pharmacological TRH-T doses and that TSH pattern is mainly due to the negative feedback of thyroid hormones, even though pituitary TSH reserves may become depleted. Furthermore, prolonged TRH-T administration does not produce hyperthyroidism in euthyroid CVD patients.     

Uptake and release of 3H prostaglandins E2 and F2 alpha in uterine strips isolated from ovariectomized rats. Influence of in vitro progesterone

The accumulation and output of 3H -prostaglandins (PGs), E2 and F2 alpha, into and from uterine strips isolated from ovariectomized rats, either in presence or in absence of exogenous progesterone, were explored. Tissue-to-medium ratio of 3H-counts (T/M-ratio), was determined. The same was done in solutions containing 14C-sucrose. During a 60 min incubation period in a solution containing 3H -PGF2 alpha, a net accumulation of radioactivity was evident in control (no progesterone) uterine slices. The T/M-ratio for 3H-PGF2 alpha, increased with time, reaching maximal values at 45 min. Progesterone (100 ug.ml-1) attenuated the uptake process, as evidenced by stable values of T/M-ratio, as time progressed. On the other hand, control T/M-ratio for 3H-PGE2, although similar to that for 3H -PGF2 alpha, was not influenced by the presence of exogenous progesterone. Regarding labelled PG release from the tissue, it was observed that, during an experimental period of 60 min, most tritium from control slices was released within the first 30 min after incubation with 3H -PGF2 alpha, whereas, following the presence and subsequent removal of exogenous progesterone, the bulk of 3H -released took place at 6-70 min. On the other hand, the release of 3H after an incubation with 3H -PGE2, was also maximal as that for 3H -PGF2, alpha within the first 30 min and resulted not altered after a period of exposure and removal of progesterone. The foregoing results suggest an specific pharmacological effect of progesterone, attenuating the uptake and retarding the outflow of PGF2 alpha, but not that of PGE2, into and from uterine slices of ovariectomized rats. Findings reported herein are discussed in terms of progesterone priming and withdrawal, in relation to PGF2 alpha fluxes in the rat uterus during the sex cycle, as well as in relation to PG binding to tissue receptors.     

Placebo-controlled study of effects of epimestrol on the pituitary-testicular axis in normal men

Twenty healthy male volunteers were randomly allocated to the treatment with either 15 mg/day of epimestrol or placebo for 10 days. The plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), oestradiol (E2) and prolactin (PRL) were measured before, during and 4 days after the medication by radioimmunoassays. Data were statistically evaluated by means of an analysis of covariance. Circulating LH and FSH, and also T and E2 significantly increased in the epimestrol treated subjects. In the placebo treated subjects no significant changes in the plasma hormone levels were observed. There were no significant changes in the plasma levels of PRL in either group.