Martha Medina

Effects of hydrazine derivatives on vascular smooth muscle contractility, blood pressure and cGMP production in rats: comparison with hydralazine.

Hydralazine is a hydrazine derivative used clinically as a vasodilator and antihypertensive agent. Despite numerous studies with the drug, its mechanism of action has remained unknown; guanylate cyclase activation and release of endothelial relaxing factors are thought to be involved in its vasodilator effect. Other hydrazine derivatives are known to stimulate guanylate cyclase and could therefore share the vasodilator activity of hydralazine, although such possibility has not been assessed systematically. In the present study, hydralazine, hydrazine, phenylhydrazine, and isoniazid were evaluated for vascular smooth muscle relaxation in rat aortic rings with and without endothelium, as well as after incubation with the guanylate cyclase inhibitor methylene blue. They were also tested for enhancement of cyclic guanosine monophosphate (cGMP) production by cultured rat aortic smooth muscle cells and for hypotension in the anesthetized rat. All hydrazines relaxed aortic rings, an action unaffected by endothelium removal and, in all cases except hydralazine, antagonized by methylene blue. Only phenylhydrazine increased cGMP production and only hydralazine markedly lowered blood pressure. It was concluded that hydralazine vascular relaxation is independent of endothelium and is not related to guanylate cyclase activation. The other hydrazines studied also elicit endothelium-independent relaxation, but the effect is related to guanylate cyclase. The marked hypotensive effect of hydralazine contrasts with its modest relaxant activity and is not shared by the other hydrazines. The fact that hydrazine and isoniazid produce methylene blue-sensitive relaxation, yet do not enhance cGMP production suggests the need for activating factors present in aortic rings but not in isolated cells.

Clinical and endocrine spectrum in patients with the 45,X/46,XY karyotype.

SummaryCytogenetic and endocrine studies were performed in five unrelated 45,X/46,XY individuals in an attempt to correlate them with their clinical expression and gonadal morphology. A lack of a consistent pattern between cytogenetic findings and phenotype was observed.Endocrine studies revealed a wide spectrum of hypothalamic, pituitary, and gonadal hormone production as assessed by the base line levels of LH, FSH, T, and Δ4-A and their responses to appropriate exogenous stimulation (LH-RH and HCG). An adequate correlation between endocrine findings with gonadal morphology and phenotype could be established; thus demonstrating that patients with this particular chromosome complement have a functional integrity of the gonadotropin hypothalamic pituitary activity modulated accordingly with the gonadal function of each particular case.

Enhancement of hydralazine hypotension by low doses of isoniazid. possible role of semicarbazide-sensitive amine oxidase inhibition

The influence of pretreatment with 1 through 300 mg/kg ip of isoniazid (ISO) on blood pressure and heart rate responses to 0.1 mg/kg iv of hydralazine (HYD) was assessed in rats anesthetized with chloralose--urethane. HYD hypotension was significantly enhanced by ISO at doses between 3 and 300 mg/kg ip. Heart rate was not influenced by HYD in control or pretreated animals. Depressor responses to 0.2 mg/kg iv of pinacidil (PIN) were also potentiated by ISO at 100 and 300, but not at 30 mg/kg. Similarly, ISO decreased cerebral gamma-aminobutyric acid (GABA) at the two highest doses; 30 mg/kg was without effect. Pretreatment of rats with ISO at 1 through 300 mg/kg failed to influence HYD-induced relaxation of aortic rings. These results were interpreted as indicating that potentiation of HYD hypotension by high doses of ISO is not specific for that vasodilator and is related to decreased cerebral GABA, as postulated previously. Lower doses could specifically potentiate the HYD-induced hypotensive effect by inhibition of semicarbazide-sensitive amine oxidase (SSAO), since both ISO and HYD are potent inhibitors of this enzyme. In support of this hypothesis, the SSAO inhibitors, benserazide (100 mg/kg ip) and mexiletine (50 mg/kg ip), were also found to enhance HYD hypotension.

Bilateral Sequential Common Carotid Artery Sectioning in Mice as a New Model for Testing Neuroprotective Drugs

The consequences of cerebral global ischemia induced by right common carotid artery (CCA) sectioning in mice previously subjected to a chronic deficiency of brain blood supply by sectioning of the contralateral artery, as well as the neuroprotector efficacy of 3 5-hydroxytryptamine 1A receptor agonists, were investigated in this study. Left CCA sectioning produced a remarkable increase in the diameter of arteries constituting the cerebral arterial circle; the effect was maximal at 32 days. No neurological deficit or significant brain damage were found in these animals. In mice with previous left CCA sectioning, sectioning of the contralateral artery gave rise to a wide range of motor-behavioral abnormalities, characteristic brain damage, and high mortality rate. Neurological abnormalities ranged from alterations in locomotor activity to lack of coordination, hemiparesis, circling, immobility, and unresponsiveness. Seventeen percent of the animals died within 15 minutes after the acute ischemic event, and the number of deaths progressively increased to 63 at 24 hours and 92 at day 8. In mice killed 72 hours after right artery sectioning, the incidence of infarction was 100, and was more pronounced in the right hemisphere. The 3 5-hydroxytryptamine 1A receptor agonists, tested reduced the severity of neurological alterations and decreased the cumulative mortality rate of the mice. The activity of ±8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide and indorenate was dose-dependent, whereas the protective action of buspirone was more pronounced with lower and higher doses than with intermediate ones. Maximal neuroprotection was obtained with ±8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide. This new model of global ischemia may be relevant to stroke in humans. It incorporates the idea of provoking an acute ischemic insult in animals subjected to chronic hypoperfusion and uses neurological and mortality endpoints to determine the effects of brain ischemia and the degree of neuroprotection. The present findings warrant continued efforts to refine our model to better reflect human stroke and to determine its usefulness in predicting the effectiveness of a particular treatment strategy.

The Stein-Leventhal Syndrome: a Neuropituitary Disorder?*

Hypothalamic and pituitary gonadotropin function and responsiveness in four patients with well-documented Stein-Leventhal syndrome were studied. All patients were of reproductive age and had had menstrual disorders since menarche. Estrogen production was assessed by measuring the circulating levels of immunoreactive estradiol, by vaginal smears, and by the progestogen-induced menses test. Gonadotropin function was evaluated by measuring the serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in blood samples obtained at 15-minute intervals for 150 minutes. Pituitary gonadotropin reserve and responsiveness were studied by giving an intravenous bolus of synthetic LH-releasing hormone (LH-RH) and measuring the circulating gonadotropin levels before and after the injection.

Proliferative Properties of 17β‐aminoestrogens in MCF‐7 Human Breast Cancer Cells

The 17β‐aminoestrogens (AEs) prolame, butolame and pentolame are weakly oestrogenic in rodents and display anticoagulant properties in contrast to oestradiol (E2) which presents pro‐coagulant effects, potentially thrombogenic. They possess anti‐anxiety and antidepressive properties, being good candidates for menopausal hormone therapy (MHT). Their capability to induce proliferation of MCF‐7 human breast cancer cells, in which proliferative rate depends on oestrogens, has not been explored; thus, the aim of this work was to characterize it. AEs’ proliferation properties were evaluated compared with E2 in MCF‐7 carcinoma cell line cultures using established methods. Receptor mediation on cell proliferation was studied by co‐incubating them with the oestrogen receptor antagonists tamoxifen, ICI 182,780 and the selective antagonists MPP (ERα) and PHTPP (ERβ). E2 and AEs increased MCF‐7 cell proliferation; their proliferative effect was between 1.5‐2 and E2 = 3.6 compared with controls (0); their relative proliferative effect was 18–38% (E2 = 100%) with a relative proliferative potency of 4.5–8.9 (E2 = 100). The ERα antagonist MPP inhibited the MCF‐7 cell proliferation induced by E2 and AEs; on the contrary, the ERβ antagonist PHTPP exacerbated the proliferative response, showing that the AEs’ proliferative activity was mainly ERα‐mediated and apparently controlled by ERβ. Preliminary cytometric DNA flow analysis showed that AEs’ cell cycle S phase inducer property was lower than E2 following the proliferative order: E2 > butolame > prolame > pentolame, indicating pentolame with the weakest proliferative properties and being the safest of this series as a candidate for MHT.

Ovariectomy differential influence on some hemostatic markers of mice and rats.

Rodent ovariectomy is an experimental method to eliminate the main source of sexual steroids. This work explored for the first time the ovariectomy temporal changes induced in the hemostatic coagulation markers: prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration (FIB) along with uterine weight on adult female CD1 mice and Wistar rats. Uterine weight (Uw) was assessed before ovariectomy (control), and 1, 3, 5, 7, 9, 16, and 21 days after surgery. PT, aPTT, TT and FIB were estimated the same days, using reported standard techniques. Ovariectomy decreased Uw, since day 1; and from day 10 to 21 reached the lowest values for both species. After day 1, mice hemostatic parameters changed (PT +10%, P<0.05; aPTT +53%, P<0.05; TT −24%, P<0.05; FIB +67%, P<0.05). Rats showed significant changes only in TT and FIB (TT −13%, P<0.001; FIB +65%, P<0.001). Neither mice PT, aPTT and TT, recovered control values after 21 days. In the rats from day 5 to 16 aPTT diminished (18–23%, P<0.05) recovering to control values on day 21, TT after 9 days and PT on day 16. In both species, FIB returned to its control values after 9 days. Ovariectomy differentially altered the PT hemostatic parameter of mice and rats indicating a non-equivalence among both species behaviour for experimental studies of blood coagulation.

In vivo profile of the anticoagulant effect of 17ß-amino-1,3,5(10)estratrien-3-ol

The anticoagulant activity of 17ß-amino-1,3,5(10)estratrien-3-ol (AE(2)) was established for the first time. Experiment 1: mice groups were treated with a single subcutaneous (s.c.) AE(2) injection (0.5, 1, 2, 4, and 8 mg/100 g BW) or vehicle (propylenglycol; 0.5 ml/100 g). After 24 h, AE(2) produced dose-dependent blood clotting time increases related to control, Emax=+121% (P<0.01) finishing the sixth day. Experiment 2: four groups received a single s.c. administration of AE(2) (4 or 8 mg/100g BW) or 17ß-estradiol (E(2); 3mg/100g BW) or vehicle. After 24 and 48 h post-administration, the times of blood clotting, prothrombin, thrombin, and activated partial thromboplastin and fibrinogen concentrations were assessed. Both AE(2) doses increased blood clotting and fibrinogen similarly, blood clotting time: 64, 94%; fibrinogen: 71, 107% (P<0.01). Prothrombin, activated partial thromboplastin and thrombin times, increased 13-15%, 27-55%, and 15-29%, respectively (P<0.01). Meanwhile E(2) decreased blood clotting 20% (P<0.01) and thrombin 23% (P<0.01) after 48 h. Experiment 3: for five consecutive days, mice received AE(2) or E(2) (0.1, 1, 10, 100, and 1000 μg/kg/day), or vehicle. Blood clotting time was assessed at 1, 2, 3, 4, 5, 8, and 11 days after treatment. AE(2) at all doses were anticoagulant for 2-3 days after administration whereas E(2) was procoagulant for 8-11 days. These opposite effects were: AE(2) Emax=+29%; E(2) Emax=-30%; (P<0.01). AE(2) is the parent compound of the 17ß-aminoestrogens, with the largest and longest anticoagulant effect until now reported.

Anticholinergic properties of progesterone in the isolated ileum of the guinea-pig

To determine if the cholinergic system is involved in the intestinal smooth muscle relaxant action of progesterone, segments of guinea‐pig ileum were mounted in 20 ml chambers containing Krebs solution. In one group of experiments, we studied the effect of various doses of progesterone and atropine on the twitch‐like contraction of the ileum caused by electrical stimulation. In the second group of experiments, dose‐response curves were constructed for acetylcholine, histamine, and barium chloride alone and in the presence of progesterone or atropine. Progesterone, like atropine, decreased in a dose‐dependent manner the phasic contractions and tone of the unstimulated intestinal segments, and exerted a rapid, reversible, and dose‐dependent inhibitory action on intestinal responses to electrical stimulation. In addition, in the presence of the hormone or atropine, the dose‐response curve to acetylcholine was clearly shifted to the right in a parallel manner with no change in maximal response. At the concentrations tested, neither progesterone nor atropine blocked the contractile effect of barium chloride on the ileum and only slightly reduced the spasmogenic action of histamine. These results suggest that the relaxant action of progesterone on the intestinal smooth muscle may be mediated through muscarinic receptors, indicating that progesterone is an endogenous product with anticholinergic activity.

In vivo and in vitro evaluation of the estrogenic properties of the 17β-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17β-estradiol

BACKGROUND Buame [17β-(butylamino)-1,3,5(10)-estratrien-3-ol] possesses anticoagulant and antiplatelet activities that are potentially antithrombotic. Since its estrogenicity is unknown, it was evaluated by established methods. METHODS Buame (10, 100, 500, and 1,000 μg/kg), 17β-estradiol (E(2)) (100 μg/kg), or propylene glycol (10 ml/kg) were subcutaneously (sc) administered for three days to immature Wistar female rats (21 days old). The relative uterotrophic effect to E(2) was 78 (E(2) = 100) with a relative uterotrophic potency of 1.48 (E(2) = 100). Adult ovariectomized Wistar rats received an sc injection at 8:00 h (reversed cycle) of: 7.5 μg of E(2) (≈ 30 μg/kg), buame (≈ 750, 1,500, 3,000 μg/kg), or corn oil (≈ 1.2 ml/kg). After 24 h, progesterone (4-5 mg/kg) was administered. Sexual receptivity was assessed 5 to 7 h later, and the lordosis quotient (LQ; number lordosis/number mounts x 100) was evaluated. RESULTS Buame induced lordosis (LQmax 85 ± 9; ED50 952 ± 19 μg/kg) and E(2) LQmax 56 ± 8; ED50 10 ± 2 μg/kg; the relative LQ-potency was 0.51 (E(2) = 100). Buame competed with [(3)H]E(2) for the estrogen receptor (Buame RBA= 0.15 and Ki = 5.9 x 10(-7) M; E(2) RBA= 100;Ki = 6.6 x 10(-9) M). Buame increased MCF-7 cells proliferation, from 10(-11) to 10(-)9 M, its proliferative effect was 1.73-1.79 (E(2) = 3.0-3.9); its relative proliferative effect to E(2) was 33-40% (E(2) = 100%) and relative potency 10.4-10.7 (E(2) = 100). Tamoxifen and fulvestrant (ICI 182,780) inhibited buames proliferation indicating mediation through estrogen receptors in this response. CONCLUSION Buame is therefore an estrogen partial agonist with a weak estrogenic activity.