Blockade of alpha1-adrenoceptors and cardiac depressant effect by a newly synthetic antihypertensive drug, DL-017 of quinazoline derivative.

The electromechanical effects of 3-[[4-(2-methoxy phenyl)piperazin-1-yl]methyl]-5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (DL-017), a newly synthesized quinazoline-derived antihypertensive agent, on mammalian cardiac tissues were evaluated. In driven canine Purkinje fibers, DL-017 decreased twitch tension, the maximal rate of upstroke of the action potential (Vmax), and intracellular Na+ activity (a(i)Na) in a concentration-dependent manner. The action potential duration was decreased in canine Purkinje fibers but increased in guinea pig papillary muscles. In guinea pig ventricular papillary muscles, phenylephrine in the presence of 1 microM propranolol increased the twitch tension in a concentration-dependent manner. At 10 microM, phenylephrine significantly decreased a(i)Na and shortened the action potential duration. DL-017 at 0.01 microM inhibited these phenylephrine-induced effects and shifted the concentration-dependent curve to the right. In sinoatrial nodes, DL-017 inhibited pacemaker activity, involving decreases in the slope of diastolic depolarization and Vmax and an increase in a delay of repolarization. These results suggest that, in addition to blockade of alpha1-adrenoceptors and Na+ channels, DL-017 reduces cardiac excitability and contractility in association with inhibition of slow inward Ca2+ and outward K+ channels. Since two order higher concentrations are required, the contribution of DL-017 to cardiac depressant from blockade of ionic channels seems to be less important when this compound is clinically used as an antihypertensive drug.

Pituitary adenylate cyclase-activating polypeptide acts synergistically with relaxin in modulating ovarian cell function in rats.

The interactive effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and relaxin on the secretion of gelatinases, involved in matrix remodeling, in ovarian theca-interstitial cells and granulosa cells, were investigated in gonadotropin-primed immature rats. The gelatinases secreted from cultured cells were analyzed using gelatin zymography and scanning densitometry. We have previously shown that relaxin stimulated the secretion of a 71 kDa gelatinase, identified as a type IV collagenase (matrix metalloproteinase 2), in rat theca-interstitial cells. This study has demonstrated that PACAP27 and PACAP38, with similar potency, dose-dependently enhanced relaxin-induced secretion of 71 kDa gelatinase, whereas PACAP alone had no effect. In rat granulosa cells, both PACAP27 and PACAP38 alone dose-dependently increased the secretion of a 63 kDa gelatinase. In addition, this study has shown that cAMP signaling pathway mediators act similarly to that of PACAP on gelatinase secretion in rat ovarian cells. Cholera toxin, forskolin and 8-bromoadenosine cAMP augmented relaxin-induced secretion of 71 kDa gelatinase in theca-interstitial cells, and alone they had no effect. These mediators also increased the secretion of 63 kDa gelatinase in granulosa cells. It is well known that the increase in cellular cAMP level is associated with the morphological rounding-up phenomenon in granulosa cells. This study has shown that PACAP and cAMP pathway mediators, but not relaxin, could cause such changes in cell shape in granulosa cells as well as in theca-interstitial cells. In conclusion, this study provides original findings that PACAP acts synergistically with relaxin in stimulating the secretion of gelatinases in rat ovarian theca-interstitial cells and granulosa cells. This supports the idea that relaxin and PACAP may serve as ovarian physiological mediators of gonadotropin function in facilitating the ovulatory process. In addition, PACAP appears to act through the cAMP signaling pathway to affect biological functions in ovarian cells, whereas relaxin does not.

TGFbeta1 stimulates the secretion of matrix metalloproteinase 2 (MMP2) and the invasive behavior in human ovarian cancer cells, which is suppressed by MMP inhibitor BB3103.

The present study investigated the modulatory role of transforming growth factor beta 1 (TGFbeta1) on the secretion of matrix metalloproteinases (MMPs) and tested whether the altered secretion of MMPs could directly affect the invasive behavior of ovarian cancer cells. To this aim, human ovarian cancer SKOV3 cells were treated once with vehicle or various concentrations of TGFbeta1 for 24 h. Gelatinase activities in conditioned media were analyzed by zymography and densitometry. TGFbeta1 dose-dependently stimulated the secretion of a 68-kDa gelatinase, which was characterized as an MMP because its activity was inhibited by a metalloproteinase inhibitor 1,10-phenanthroline, and by a synthetic MMP inhibitor BB3103. In addition, we used aminophenylmercuric acetate (APMA) to activate latent gelatinases. APMA time-dependently decreased the activity of 68-kDa gelatinase, and increased the activities of 64- and 62-kDa gelatinolytic bands. The 68-kDa gelatinase was further characterized as MMP2 (gelatinase A) by immunoblotting analysis. We then tested TGFbeta1 effect on the invasive potential of SKOV3 cells as assessed by the migration ability through reconstituted basement membrane, and further investigated whether TGFbeta1 may act through modulating the MMP activity to affect ovarian cancer cell invasion. The results show that TGFbeta1 stimulated the invasive behavior of SKOV3 cells, and that MMP inhibitor BB3103 abrogated this effect of TGFbeta1. In conclusion, this study indicates that TGFbeta1 may act partly through stimulating the secretion of MMP in promoting the invasive behavior of human ovarian cancer cells. Furthermore, this work supports the idea that specific MMP inhibitors of the hydroxamate class could be therapeutically useful in controlling cancer cell invasion/metastasis.

Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis-associated properties in A431 cells overexpressing epidermal growth factor receptor.

1. Flavonoids display a wide range of pharmacological properties including anti-inflammatory. Anti-mutagenic, anti-carcinogenic and anti-cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion. 2. Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 micronM, respectively. 3. The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25+/-4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 micronM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR. 4. A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time-dependently induced the appearance of a ladder pattern of DNA fragmentation, and this effect was abolished by EGF treatment. 5. The addition of EGF only marginally diminished the inhibitory effect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase. 6. EGF increased the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells. 7. Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3' and C4' in ring B are critical for the biological activities. 8. This study demonstrates that the inhibitory effects of Lu and Qu, and the stimulatory effects of EGF, on tumour cell proliferation, cellular protein phosphorylation, and MMP secretion may be mediated at least partly through EGFR. This study supports the idea that Lu and Qu may have potential as anti-cancer and anti-metastasis agents.

Relaxin modulates the ovulatory process and increases secretion of different gelatinases from granulosa and theca-interstitial cells in rats.

There were two related objectives in this study. The first was to determine the influence of endogenous relaxin on ovulation in rats. The second was to investigate the effect of relaxin on the secretion of gelatinases involved in extracellular matrix remodeling from rat ovarian cells. Immature rats were primed s.c. with 10 IU eCG; 51 to 52 h later, a monoclonal antibody specific for rat relaxin (MCAR), a control antibody against fluorescein (MCAF), or PBS vehicle was administered via intraovarian bursal injection under anesthesia, and 15 IU hCG was injected i.p. immediately thereafter. Rats were killed 26 h later, and oviducts were isolated and examined under the microscope to determine the number of ovulated oocytes. MCAR (0.25 and 2.5 micrograms/ovary) partially suppressed gonadotropin-induced ovulation as compared to the value for PBS controls. There was no significant difference in the number of ovulated oocytes between animals treated with MCAF and PBS controls. Also, porcine relaxin, given s.c. immediately after MCAR treatment, could reverse the inhibitory effect of MCAR on ovulation. To examine a possible mechanism for the effect of relaxin on ovulation, granulosa cells and theca-interstitial cells were obtained from ovaries of eCG-primed immature rats. The gelatinases secreted from cultured cells were analyzed using gelatin zymography and scanning densitometry. In the granulosa cell culture, relaxin increased the secretion of two major gelatinases of about 92 and 63 kDa in a dose-and time-dependent manner within 24 h of treatment. In the theca-interstitial cell culture, relaxin induced dose- and time-dependent increases in the secretion of two other major gelatinases of about 76 and 71 kDa. These gelatinases were characterized as metalloproteinases but not serine/cysteine proteinases. Furthermore, an immunoblot study demonstrated that relaxin stimulated the secretion of a 72-kDa type IV collagenase-like substance from cultured theca-interstitial cells but not from granulosa cells. This study demonstrates several original findings. First, endogenous relaxin may facilitate the ovulatory process in rats. Second, exogenous relaxin exhibits a biological effect on cultured rat theca-interstitial cells in addition to granulosa cells. Third, exogenous relaxin regulates the secretion of different major forms of gelantinases from cultured rat granulosa cells and theca-interstitial cells. The study supports the idea that relaxin may play an autocrine/paracrine role that is involved in modulating ovarian function.

Signal transduction mediated by angiotensin II receptor subtypes expressed in rat renal mesangial cells.

Recent evidence suggests that there are two classes of receptors for angiotensin II (AngII), AT1 which is sensitive to losartan (DuP753) and is G-protein coupled, and AT2 which is sensitive to both PD123319 and CGP42112A, and is non-G-protein coupled. In rat mesangial cells two subtypes of AT1 receptor could be distinguished, AT1A subtype is more sensitive to losartan whereas AT1B subtype is more sensitive to PD123319, but insensitive to CGP42112A. The present studies were designed to ascertain which receptor subtype mediates three AngII-induced physiologic functions in rat mesangial cells namely intracellular Ca2+ mobilization, adenylyl cyclase inhibition and protein synthesis as monitored via [3H]leucine incorporation. The rank order of potency for inhibition of AngII-induced [Ca(2+)]i mobilization and adenylyl cyclase regulation was PD123319 > or = losartan > CGP42112A. By contrast, losartan was quite effective at inhibiting protein synthesis (IC50 = 8 nM) while PD123319 was without effect. These findings are consistent with AngII mediated signal transduction through AT1A and AT1B sites for phospholipase C mediated [Ca(2+)]i mobilization and inhibition of adenylyl cyclase. On the other hand, AT1A receptors appear to exclusively mediate AngII-induced protein synthesis. These observations underscore the complexity of AngII mediated signal transduction in glomerular mesangium.

Binding of progesterone to nerve cell membranes of rat brain using progesterone conjugated to 125I-bovine serum albumin as a ligand.

Radioiodinated bovine serum albumin conjugated to progesterone was used as a probe to examine binding parameters of steroids to membrane preparations from rat brain tissue. The binding of 11 alpha-hydroxyprogesterone-11-hemisuccinate-125I-bovine serum albumin conjugate reached saturation after 30 min of incubation at 5 degrees C. Several bovine serum albumin-conjugated steroids were then tested for competition displacement studies. Among these steroid conjugates, the bovine serum albumin conjugate at position 3 of progesterone had the highest affinity, with an estimated inhibition constant of 28.5 +/- 2.1 nM (n = 3), whereas bovine serum albumin itself and the 17 beta-estradiol 6-(O-carboxy-methyl)oxime-bovine serum albumin conjugate showed no specific displacement. In addition, the binding sites were localized in an axolemma-enriched fraction of rat brainstem. Specific binding was obtained in tissues from cerebral cortex, brainstem, cerebellum, corpus striatum, and hypothalamus, but little or no binding occurred in uterus, ovary, liver, and spleen. The present data indicate that progesterone-125I-bovine serum albumin conjugate can be used as a ligand to study progesterone-membrane receptor interactions.

Effects of progesterone and its metabolites on neuronal membranes.

Evidence supporting a membrane site of action for progesterone includes the rapidity of its effects when directly infused into tissue containing mainly nerve terminals, the absence of functional intracellular progesterone receptors in vitro and the fact that progesterone conjugated to bovine serum albumin (BSA) in the C-3 position (P-3-BSA) activates the release of hypothalamic luteinizing hormone releasing hormone (LHRH) or modulates amphetamine-evoked striatal dopamine release. In addition, P2 membrane fractions from different areas of the CNS but not P1 fractions or P2 membranes from peripheral progesterone targets have specific binding sites for P-11-125I-BSA. Among several BSA-conjugated steroids tested for competition displacement P-3-BSA had the highest affinity with an estimated inhibition constant of 28.5 +/- 2.1 nM. This binding depends on the presence of cations such as Ca2+ and Mg2+ and after chemical depolarization of the P2 membranes the binding curve of P-3-BSA shifts to the right. While progesterone is effective in releasing LHRH from the hypothalamus, 5 beta-pregnan-3 beta-ol-20-one (a 5 beta reduced metabolite) is at least 1000-fold more potent than the parent compound when tested in vitro and in vivo. This action is indirect because tetrodotoxin at 10(-6) M blocks the LHRH releasing action, although 5 beta-prenan-3 beta-ol-20-one is still capable of releasing noradrenaline. Although 5 beta-pregnan-3 beta-ol-20-one can replace progesterone in activating the LHRH neural apparatus this is not true for the nigro-striatal dopamine system where only progesterone or P-3-BSA is effective, an action which is also indirect since tetrodotoxin blocks the effect of either compound. These results indicate that progesterone acts at membrane sites to modulate specific functions of the CNS and that site-specific mechanisms exist within the CNS which may differentially control its conversion to more active compounds.

Membrane mechanism mediates progesterone stimulatory effect on LHRH release from superfused rat hypothalami in vitro.

To determine whether the plasma membrane is a primary site for progesterone (P4) action on the neural LHRH apparatus of hypothalamic tissues derived from ovariectomized, estradiol-primed (OVX + E2) immature rats, immobilized P4 was infused directly to these tissues using a superfusion technique. Two kinds of immobilized P4 with bovine serum albumin (BSA) conjugated at positions 3 or 11, or 11-deoxycorticosterone (DOC) immobilized at position 21 of the steroid molecule, respectively, were tested for structural specificity. Among the three immobilized steroids, only P4 with BSA conjugated at position 3 (P4-3-BSA) was effective in stimulating LHRH release in vitro. P4-3-BSA at 0.5 micrograms/ml, approximately 1.7 X 10(-7) M of P4, increased LHRH levels in the superfusates to about 2.5-fold those of pretreatment levels. In addition, no conversion of P4-3-BSA to free progesterone was detected. This observation demonstrated that the plasma membrane is a primary site for the stimulating effect of P4 on LHRH release from hypothalamic tissue in vitro.

High-pressure liquid chromatographic separation of a mixture of corticosteroids, androgens, and progestins.

Fifteen steroids including corticosteroids, androgens, progestins, and their derivatives were completely separated by reverse-phase high-pressure liquid chromatography on a ChemcoPak 7 ODS-H column in 50 min. The elution procedures were first with water:methanol:acetonitrile:isopropanol 55:32:6.5:7.5 (v/v) for 15 min and followed with a linear gradient elution for 35 min from 0 to 80% of water:methanol:n-butanol 40:40:20 (v/v). The applicability of this method was successfully demonstrated in the analyses of the biological samples of carp plasma, testis, and head kidney.