Hwee Teoh

Differential effects of 17β-estradiol and testosterone on the contractile responses of porcine coronary arteries

We investigated the effects of short‐term exposure to physiological levels of 17β‐estradiol and testosterone on vasocontractile responses in porcine coronary artery rings. Concentration‐response curves to endothelin‐1, 5‐hydroxytryptamine, the thromboxane analogue U46619 and KCl were constructed in endothelium‐intact and endothelium‐disrupted artery rings. Thirty minutes exposure to 17β‐estradiol (1 and 30u2003nM) significantly attenuated vasoconstriction to endothelin‐1, 5‐hydroxytryptamine and U46619. Conversely, the same concentrations of testosterone significantly potentiated responses elicited by these contractile agents. These inhibitory effects of 17β‐estradiol and enhancing actions of testosterone on contractions were endothelium‐independent. KCl‐mediated contractions were unaffected by the presence of either sex hormones. The oestrogen receptor antagonists, tamoxifen (10u2003μM) and ICI 182,780 (10u2003μM), were unable to reverse the inhibitory influence 1u2003nM 17β‐estradiol had on the agonist‐mediated contractile responses. Similarly, the androgen receptor antagonists, flutamide (10u2003μM) and cyproterone acetate (10u2003μM), failed to affect the potentiating activities of 1u2003nM testosterone. The alteration in vasoconstrictive responses observed following acute exposure to either 1u2003nM 17β‐estradiol and 1u2003nM testosterone were apparent even in the presence of the protein synthesis inhibitor cycloheximide (10u2003μM) and the transcription inhibitor actinomycin D (10u2003μM). In conclusion, we report a unique type of sex hormone action on the coronary vasculature. These events occur at low nanomolar concentrations of 17β‐estradiol and testosterone, are insensitive to conventional sex hormone receptor antagonists, are not blocked by de novo protein synthesis inhibitors and have rapid time‐courses that are uncharacteristic of classical genomic activities.

GABA, glutamate and substance P‐like immunoreactivity release: effects of novel GABAB antagonists

1 The effects of various GABA receptor ligands on the electrically‐evoked release of endogenous GABA, glutamate and substance P‐like immunoreactivity from the dorsal horn of rat isolated spinal cord were examined. 2 Exogenous GABA (10–300 μm) significantly decreased the evoked, but not basal, release of endogenous glutamate in a concentration‐dependent manner. The GABAA agonist, isoguvacine (1–100 μm), failed to decrease the release of glutamate although it did reduce the release of GABA. Baclofen (0.1–1000 μm), the GABAB agonist, reduced the release of GABA and glutamate in a stereospecific and concentration‐dependent manner. 3 The actions of five GABAB antagonists on these release systems were compared. CGP36742, CGP52432, CGP55845A and CGP57250A significantly increased the evoked release of GABA and glutamate. They also reversed the effects of (−)−baclofen in a concentration‐dependent manner. On the other hand, while CGP56999A had no effect on glutamate release, it was an effective antagonist of the baclofen‐induced inhibition of GABA and substance P release. 4 These results suggest that GABAB receptors on nerve terminals within the dorsal horn spinal cord may be heterogeneous. However, this is based solely on the data obtained with CGP56999A which affected only GABA and substance P, but not glutamate, release.

Short-term exposure to physiological levels of 17β-estradiol enhances endothelium-independent relaxation in porcine coronary artery

Objectives: While alterations in cholesterol and lipoprotein profiles partly account for menopause being a risk factor for coronary heart disease, recent studies have suggested that 17β-estradiol may have vascular effects. Our aims were to study the short-term effects of 17β-estradiol on vascular function in isolated porcine coronary artery rings. Concomitantly, we sought to determine if physiological concentrations of 17β-estradiol could acutely potentiate relaxation. Results: 17α- and 17β-estradiol at pharmacological (>1 μM) concentrations produced relaxation in U46619-pre-contracted porcine coronary artery rings. Relaxation evoked by 17β-estradiol was not reversed by the estrogen receptor antagonists tamoxifen and ICI 182780. Following 20 min exposure to a physiological concentration of 17β-estradiol (1 nM), which on its own had no effect, relaxation elicited by cromakalim, levcromakalim and sodium nitroprusside, but not bradykinin or calcium ionophore A23187, were significantly enhanced. This potentiating action was also insensitive to tamoxifen and ICI 182780. Our data provide evidence for an acute indirect relaxant action of 17β-estradiol and suggest that it may be via a tamoxifen- and ICI 182780-insensitive estrogen receptor. While this response was only observed at pharmacological concentrations, the potentiation of cromakalim, levcromakalim and sodium nitroprusside relaxation was evident in the presence of a physiological concentration (1 nM) of 17β-estradiol. Conclusions: These results demonstrate that short-term exposure to 17β-estradiol, at concentrations that have no effect on their own, can enhance vasorelaxation. These vascular effects may partly account for some of the acute effects of 17β-estradiol on blood flow.

Panax quinquefolium saponins protects low density lipoproteins from oxidation

Oxidized low-density lipoprotein (Ox-LDL) is believed to be involved in the pathogenesis of atherosclerosis. Panax quinquefolium saponins (PQS) are extracted from the stems and leaves of the North American form of ginseng, Panax quinquefolium. Earlier studies have suggested that this extract improves the lipid profile of hyperlipidemic rats and has antioxidant properties in cultured rat cardiac myocytes. The aims of the present study were to investigate the potential of PQS in reducing LDL oxidation as well as limiting the ability of Ox-LDL to impair endothelium-dependent relaxation in rat aortic rings. LDL was isolated from the plasma of healthy human donors by sequential ultracentrifugation. Native LDL (0.2 or 0.3 mg/ml) was incubated with PQS (0.25-1 mg/ml) for 30 min at 20 degrees C. For comparison, vitamin C (50 microM) was added in place of PQS. Oxidative modification was initiated with 5 microM CuSO4 at 37 degrees C for 0-24 h. In our hands, PQS concentration-dependently reduced lipid peroxide levels as measured by the amount of thiobarbituric acid reactive substances formed. This range of PQS also retarded the alterations in relative electrophoretic mobility of Ox-LDL in a similar manner. Furthermore, measurement of phospholipid fractions content indicated that PQS could reduce the conversion of phosphatidylcholine to lysophosphatidylcholine in Ox-LDL. Functional studies demonstrated that PQS-pretreated Ox-LDL was less potent than untreated Ox-LDL at impairing endothelium-dependent relaxation in rat aortic rings. In conclusion, our results suggest that PQS has antioxidant properties and that reduction of LDL oxidation by PQS may provide a protective effect against the detrimental actions of Ox-LDL.

Enhanced relaxation of porcine coronary arteries after acute exposure to a physiological level of 17β-estradiol involves non-genomic mechanisms and the cyclic AMP cascade

The present study extends our previous finding that the endothelium‐independent relaxation in porcine coronary artery rings is enhanced after short‐term (20u2003min) exposure to a physiological concentration (1u2003nM) of 17β‐estradiol and demonstrates that this effect may be attributable to activation of the cyclic AMP pathway. Isometric tension was recorded in isolated rings of porcine coronary arteries. Relaxation by levcromakalim and sodium nitroprusside, but not bradykinin and calcium ionophore A23187, were significantly potentiated following 20u2003min treatment with 1u2003nM 17β‐estradiol. This enhancing effect was insensitive to the transcriptional and translational inhibitors, actinomycin D and cycloheximide respectively and absent following repeated washing of the rings prior to construction of relaxation‐response curves. The potentiating actions of 1u2003nM 17β‐estradiol on endothelium‐independent relaxation were mimicked by the cyclic AMP analogue 8‐Bromo‐cyclic AMP and the protein kinase A activator Sp‐cyclic AMPS but not by the cyclic GMP analogue 8‐Bromo‐cyclic GMP. The modulatory effect of 17β‐estradiol was increased in the presence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine. The cyclic AMP‐dependent protein kinase A inhibitor Rp‐cyclic AMPS, but not the cyclic GMP antagonist Rp‐8‐Bromo‐cyclic GMPS, effectively inhibited the enhancing effects 1u2003M 17β‐estradiol had on the relaxation responses of levcromakalim and sodium nitroprusside. These data support our earlier findings that physiologically relevant concentrations of 17β‐estradiol can acutely modify vasorelaxation in vitro. Furthermore, we report that this short‐term effect of 17β‐estradiol on vasorelaxation appears to be mediated via non‐genomic pathways and involves the cyclic AMP cascade.

Acute impairment of relaxation by low levels of testosterone in porcine coronary arteries.

OBJECTIVESnWhile there are many suggested reasons for the marked gender bias in cardiovascular events, much of the available data indicate that circulating estrogens are cardioprotective. The possibility that endogenous androgens may be detrimental to the cardiovascular system has received relatively less attention. We investigated the short-term modulatory effects of various concentrations of testosterone on vascular function in isolated porcine coronary artery rings.nnnRESULTSnThe higher concentrations (> 1 microM) of testosterone relaxed U46619-contracted coronary artery rings in an endothelium-independent manner. This direct effect was insensitive to the testosterone receptor antagonists, flutamide and cyproterone acetate. Short-term exposure (20 min) to low levels of testosterone (1-100 nM), which were ineffective on their own on vascular function, significantly diminished relaxation to bradykinin and calcium ionophore A23187 but not those produced by levcromakalim and sodium nitroprusside. The inhibitory effect observed with 1 nM testosterone was only partially reversed by flutamide and cyproterone acetate and unaltered in the presence of actinomycin D and cycloheximide.nnnCONCLUSIONSnThese results demonstrate that acute treatment with testosterone, at concentrations that have no effect on their own, reduces vasorelaxation. Furthermore, they suggest that this modulatory action may be in part independent of the classical testosterone receptor since it was not completely sensitive to the anti-androgens and was not inhibited by the transcriptional and translational inhibitors. These findings support the postulation that testosterone may have unfavorable influences on vascular function.

NON-GENOMIC VASCULAR ACTIONS OF FEMALE SEX HORMONES: PHYSIOLOGICAL IMPLICATIONS AND SIGNALLING PATHWAYS

1 Epidemiological studies indicate a lower incidence of coronary heart disease in premenopausal women compared with age‐matched men and post‐menopausal women. Accumulating evidence suggests that this cardiovascular protection observed in premenopausal women is at least partially attributed to the direct action of oestrogens on the vascular system. 2 Research focused on vascular actions of 17β‐oestradiol indicates that this female sex hormone favourably modulates vascular reactivity at physiological concentrations. The vascular actions of 17β‐oestradiol appear independent of its genomic actions. Both endothelium‐dependent and ‐independent signalling cascades have been implicated in the vascular effects of 17β‐oestradiol. 3 However, clinical trials on hormone‐replacement therapy argue against a role of oestrogens in preventing the development of coronary heart disease. Supplementation with oestrogen is also complicated with the increased risk of breast and endometrial cancer. Hence, a better understanding of the vascular actions of 17β‐oestradiol will serve to enhance our understanding of its role in coronary heart disease.

Interactions between Panax quinquefolium saponins and vitamin C are observed in vitro.

Inasmuch as the oxidation of low-density lipoprotein (Ox-LDL) may play a key role in the initiation and progression of atherosclerosis, it has become increasingly important to identify potential antioxidants. Panax quinquefolium saponins (PQS) are extracted from the stems and leaves of the North American form of ginseng,Panax quinquefolium. Our previous studies have indicated that PQS (0.25-1 mg/ml) can protect against oxidation of LDLin vitro. The purpose of the current work was to investigate the potential interaction of lower concentrations of PQS (1-100 μg/ml) with vitamin C on the reduction of LDL oxidation. LDL was isolated from the plasma of healthy human donors by sequential ultracentrifugation. Native LDL (0.05 or 0.2 mg/ml) was incubated with PQS and/or vitamin C for 30 min at 20°C. Oxidative modification was initiated with 2 μM or 5 μM CuSO4 at 37°C for 0-24 h. Pretreatment with PQS (100 μg/ml) reduced alterations in phospholipids, lipid peroxide levels and relative electrophoretic mobility of Ox-LDL. The presence of vitamin C (1-10 μM) significantly enhanced the protective effects of PQS. Pretreatment with PQS (1-100 μg/ml) resulted in concentration-dependent inhibition of LDL oxidation and prolongation of lag time as determined from measurements of conjugated lipid hydroperoxide content in Ox-LDL samples. Interestingly, the inhibitory actions of lower amounts of PQS (1 and 10 μg/ml) on the formation of conjugated dienes were significantly increased when vitamin C (0.1 or 1 μM) was present. In conclusion, our results suggest that PQS not only have direct antioxidant property but at low concentrations, their actions can be enhanced by vitamin C.

Low concentrations of 17β-estradiol reduce oxidative modification of low-density lipoproteins in the presence of vitamin C and vitamin E

Abstract Micromolar concentrations of estradiol are required to inhibit the oxidation of low-density lipoproteins (LDL) in vitro. Recent evidence suggests that estradiol must be modified before it can become an effective antioxidant at physiological levels. Our aim was to determine other possible conditions under which low concentrations of 17β-estradiol can reduce LDL oxidation. LDL susceptibility to oxidation was monitored by measurements of conjugated diene formation. High levels of 17β-estradiol reduced oxidative modification of LDL. Vitamin C and vitamin E also increased LDL resistance to Cu 2+ -mediated oxidation. More importantly, 10 nM 17β-estradiol, which on its own had no effect, exhibited significant antioxidant actions in the presence of either vitamins C or E. In conclusion, supraphysiological concentrations of 17β-estradiol are required to exert antioxidant effects directly in vitro. However, in the presence of vitamins C and E, concentrations of 17β-estradiol close to physiological levels can also protect LDL from oxidation.

Progesterone modulates estradiol actions: Acute effects at physiological concentrations

The progestin element of hormone replacement therapy may reduce the cardioprotective actions of the estrogen component. Only high concentrations (microM) of progesterone directly relaxed U46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F2alpha)-pre-contracted porcine coronary artery rings. A low concentration of progesterone (1 nM), with no effects of its own, shifted the relaxation curves of bradykinin and calcium ionophore A23187 to the right while not affecting those of sodium nitroprusside and levcromakalim. The negative influence that 1 nM progesterone exerted on bradykinin- and A23187-mediated relaxation was diminished when 1 nM 17beta-estradiol was concomitantly added to the bathing medium. Conversely, the potentiating actions of 1 nM 17beta-estradiol on relaxations elicited by sodium nitroprusside and levcromakalim were reduced following simultaneous treatment with the same concentrations of progesterone. These findings represent the first evidence for an acute in vitro vascular effect of progesterone at a physiologically relevant concentration and concur with previous in vivo reports demonstrating that progesterone may diminish the beneficial effects of estrogens.