Bukhtiar H. Shah

Inhibitory effect of curcumin, a food spice from turmeric, on platelet-activating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca2+ signaling

Curcumin, a dietary spice from turmeric, is known to be anti-inflammatory, anticarcinogenic, and antithrombotic. Here, we studied the mechanism of the antiplatelet action of curcumin. We show that curcumin inhibited platelet aggregation mediated by the platelet agonists epinephrine (200 microM), ADP (4 microM), platelet-activating factor (PAF; 800 nM), collagen (20 microg/mL), and arachidonic acid (AA: 0.75 mM). Curcumin preferentially inhibited PAF- and AA-induced aggregation (IC50; 25-20 microM), whereas much higher concentrations of curcumin were required to inhibit aggregation induced by other platelet agonists. Pretreatment of platelets with curcumin resulted in inhibition of platelet aggregation induced by calcium ionophore A-23187 (IC50; 100 microM), but curcumin up to 250 microM had no inhibitory effect on aggregation induced by the protein kinase C (PKC) activator phorbol myrsitate acetate (1 microM). Curcumin (100 microM) inhibited the A-23187-induced mobilization of intracellular Ca2+ as determined by using fura-2 acetoxymethyl ester. Curcumin also inhibited the formation of thromboxane A2 (TXA2) by platelets (IC50; 70 microM). These results suggest that the curcumin-mediated preferential inhibition of PAF- and AA-induced platelet aggregation involves inhibitory effects on TXA2 synthesis and Ca2+ signaling, but without the involvement of PKC.

Studies on antihypertensive and antispasmodic activities of methanol extract of Acacia nilotica pods

A methanol extract of Acacia nilotica pods (AN) caused a dose‐dependent (3–30 mg/kg) fall in arterial blood pressure. Treatment of animals with atropine abolished the vasodilator response of acetylcholine (ACh), whereas the antihypertensive effect of the plant extract remained unaltered. Phentolamine (an α‐adrenergic blocker) abolished the vasoconstrictor effect of norepinephrine (NE), whereas pretreatment of the animal with AN, did not modify the NE response. These results indicate that the antihypertensive effect of plant extract is independent of muscarinic receptor stimulation or adrenoceptor blockade. In the in vitro studies, AN produced a dose‐dependent (0.3–3.0 mg/mL) inhibitory effect on force and rate of spontaneous contractions in guinea‐pig paired atria. Similarly, it inhibited the spontaneous contraction of rabbit jejunum in a concentration‐dependent (0.1–3.0 mg/mL) manner. AN also inhibited K+‐induced contractions in rabbit jejunum at a similar concentration range, which suggests that the antispasmodic action of AN is mediated through calcium channel blockade, and this may also be responsible for the blood pressure lowering effect of AN, observed in the in vivo studies. Copyright

The antiplatelet aggregatory activity of Acacia nilotica is due to blockade of calcium influx through membrane calcium channels

1. The extract of Acacia nilotica (A. nilotica) blocked platelet aggregation mediated by platelet agonists, arachidonic acid (0.75 mM), ADP (4.3 microM), platelet activating factor (800 nM) and collagen (638 nM) in a dose-dependent manner. 2. The extract (0.21-1.4 mg/ml) blocked the platelet aggregation induced by Ca2+ ionophore, A-23187 (6 microM), in a dose-dependent manner, indicating that the Ca2+ influx is involved in aggregation. 3. The plant extract also inhibited aggregation in platelets pretreated with phorbol, 12-myristate, 13-acetate (196 nM) alone or in combination with ADP (4.3 microM), indicating an effect on protein kinase C. 4. These results indicate that the antiplatelet aggregatory activity of the extract of A. nilotica is mainly due to blockade of Ca2+ channels, although evidence also suggests the involvement of protein kinase C.

NEONATAL ANDROGEN MANIPULATION DIFFERENTIALLY AFFECTS THE DEVELOPMENT OF MONOAMINE SYSTEMS IN RAT CEREBRAL CORTEX, AMYGDALA AND HYPOTHALAMUS

Neural tissue during perinatal life is sufficiently plastic to respond to the presence of testicular androgens. Here we studied the effect of neonatal androgen manipulation (castration of male and androgenization of female rats) on monoamine neurotransmitter systems in the cerebral cortex and sexually dimorphic regions of brain (hypothalamus and amygdala). Norepinephrine (NE) and dopamine (DA) concentrations in cortex, amygdala and hypothalamus of rats were assayed by HPLC at days 25, 60, 120, 180, 240 and 300. Results show that NE levels in all groups of rats at day 25 were higher in the hypothalamus (5-9 ng/mg protein) compared to the amygdala (0.5-3 ng/mg protein) and the cortex (0.5-1 ng/mg protein). Levels of DA at day 25 in the hypothalamus and the amygdala were comparable (up to 3.5 ng/mg) but higher than in the cortex (1.25-1.75 ng/mg protein). NE and DA concentrations in sham-castrated male and androgen-treated females were higher at day 25 compared to castrated male and control females in both amygdala and hypothalamus; however, levels of NE and DA remained unchanged in the cortex. Pattern of NE concentrations started reversing with increase in age, i.e., NE levels in control females and castrated males increased almost 4-fold in amygdala and 2-fold in hypothalamus by day 300, but there was no significant change in the cortex. Compared to that, NE levels decreased in sham-castrated male (2-fold) and androgen-treated females (3-fold) in amygdala as well as in the hypothalamus (2-fold) in both these groups. A similar pattern of reversal of DA levels was found in both amygdala and hypothalamus, however, at day 300 DA levels were comparable in all the four groups. These studies suggest that androgen manipulation (castration or androgen administration) induces age-dependent short- and long-term effects on the development of noradrenergic and dopaminergic systems in the sexually dimorphic regions of brain, amygdala and hypothalamus, without a significant change in the cerebral cortex.

Co-activation of Gi and Gq proteins exerts synergistic effect on human platelet aggregation through activation of phospholipase C and Ca2+ signalling pathways.

Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-hydroxytryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 µM) and epinephrine (0.5-2 µM) was inhibited by α2-adrenoceptor blocker (yohimbine; IC50= 0.4 µM), calcium channel blockers (verapamil and diltiazem with IC50 of 10 and 48 mM, respectively), PLC inhibitor (U73122; IC50=6 µM) and nitric oxide (NO) donor, SNAP (IC50=1.6 µM)). The data suggest that synergistic effects of platelet agonists are receptor-mediated and occur through multiple signalling pathways including the activation PLC/Ca2+ signalling cascades.

Perinatal Exposure to Morphine Disrupts Brain Norepinephrine, Ovarian Cyclicity, and Sexual Receptivity in Rats

The effect of perinatal exposure to morphine on the development of catecholaminergic and reproductive function in female rats was investigated. Adult rats received morphine intraperitoneally daily for 40 days. The dose of morphine was progressively increased at 10-day intervals from 5, 7.5, 10 to 15 mg/kg body weight until day 40. The rats were mated between days 38 and 45. Administration of morphine at dose rates of 20 and 30 mg/kg continued during pregnancy. The dose was increased to 40 mg/kg for 10 days postpartum. Results showed that morphine disrupted ovarian cyclicity in 52% of the females. Amongst the remaining females, 43% became pregnant when mated. In the female offspring born to such dams, sexual maturation was delayed and body weight was reduced until weaning. At adulthood, lordosis behavior was inhibited when the female offspring were tested against stimulus males. Plasma estradiol and ovarian estradiol and progesterone levels were reduced. Norepinephrine concentration in the hypothalamus was reduced, whereas it remained unchanged in the amygdala. Dopamine concentrations in both hypothalamus and amygdala were not influenced by perinatal morphine exposure. These results suggest that chronic morphine treatment during perinatal life selectively influences the development of noradrenergic mechanisms in the rat brain and this may in turn be responsible for reduced reproductive activity.

The inhibitory effect of cinchonine on human platelet aggregation due to blockade of calcium influx

The Cinchona bark contains alkaloids like quinine, quinidine, cinchonine and cinchonidine. These agents are effective antimalarial drugs and have been used clinically in malaria caused by Plasmodium falciparum. Previous studies show that quinine and quinidine exert effects on cardiovascular system. This study was conducted to examine the effect of cinchonine on human platelet aggregation. The results show that cinchonine inhibited platelet aggregation mediated by platelet agonists, epinephrine (200 microM), ADP (4.3 microM), platelet activating factor (PAF; 800 nM) and collagen (638 nM) but had no effect on arachidonic acid (AA; 0.75 mM). Cinchonine was most effective in inhibiting aggregation induced by platelet activating factor and epinephrine with IC50 values of 125 and 180 microM respectively, however, higher concentrations of cinchonine were required to inhibit aggregation mediated by ADP or collagen (IC50; 300 microM). Pretreatment of platelets with cinchonine inhibited aggregation caused by Ca2+ ionophore, A-23187 (6 microM), in a dose-dependent manner (IC50; 300 microM) indicating an inhibitory effect on Ca2+-signaling cascade. This was supported by measuring [Ca2+]i in platelets loaded with Fura-2AM where cinchonine inhibited the rise in cytosolic Ca2+ mediated by A-23187 (6 microM) or collagen (638 nM). Results show that cinchonine (20 microM) also inhibited aggregation when platelets were pretreated with protein kinase C (PKC) activator, phorbol myristate acetate (PMA; 0.1 microM) in combination with low doses of platelet activating factor (80 nM). Cinchonine, however, had no effect on AA-induced platelet aggregation and thromboxane A2 (TXA2) synthesis in platelets. These results suggest that antiplatelet effects of cinchonine are mediated mainly through inhibition of Ca2+-influx and protein kinase C pathways in platelets.

DUAL EFFECTS OF NIMESULIDE, A COX-2 INHIBITOR, IN HUMAN PLATELETS

Nimesulide (CAS 51803-78-2) has been shown to exert marked anti-inflammatory effect in several in vivo models of inflammation. Since nimesulide is considered to be a selective inhibitor of COX-2, it has not been studied in detail in relation to its mechanistic effects on platelets, which express COX-1. This study was conducted to investigate the effects of nimesulide in platelet aggregation. We show that nimesulide (1-100 microM) inhibited platelet aggregation induced by adrenaline (20-200 microM). It also inhibited thromboxane A2 (TXA2) formation by platelets at low concentration (IC50; 1 microM). However, much lower concentrations of nimesulide (0.01-0.1 microM) potentiated the aggregatory response of subthreshold concentrations of adrenaline (0.2-2 microM). Such an effect was blocked by Ca2+-channel blockers, verapamil and diltiazem (IC50: 7 and 46 microM, respectively), nitric oxide donor, SNAP (IC50; 2 microM) and cinchonine (10 nM) but not by genistein (up to 10 microM). These results are indicative of the concentration-dependent dual effects of nimesulide on human platelet aggregation. The synergistic effect of low doses of nimesulide and adrenaline seems to be mediated through inhibition of multiple signalling pathways.

Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine.

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 µM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Agonist‐dependent differential effects of berberine in human platelet aggregation

This study was conducted to investigate the antiplatelet effects of berberine, an alkaloid found in Berberis aristata and some other plants. Platelet rich plasma, prepared from blood of normal human volunteers, was treated with varying concentrations of berberine, and the platelet aggregation in response to various agonists was measured by a Lumi‐aggregometer. The results show that berberine selectively inhibited platelet aggregation induced by collagen (638 nM) in a dose‐dependent manner (1–100 μM). In contrast, low doses of berberine (up to 30 μM) potentiated the ADP (4 μM)‐induced aggregation. The aggregation induced by other platelet agonists, AA (1.7 mM), adrenaline (20 μM), calcium ionophore, A23187 (1 μM) and PAF (800 nM) was not affected by berberine at a dose range of 1–100 μM. These results are indicative that berberine selectively inhibits collagen‐induced platelet aggregation. Since berberine was unable to inhibit the aggregation mediated by activation of thromboxane A2, increase in calcium influx, or stimulation of G‐protein linked pathways, it is likely that berberine selectively inhibits platelet aggregation by interfering with the collagen‐mediated adhesion process.