Angela M. LeDay

Safety and mechanism of appetite suppression by a novel hydroxycitric acid extract (HCA-SX)

A growing body of evidence demonstrates the efficacy of Garcinia cambogia-derived natural (–)-hydroxycitric acid (HCA) in weight management by curbing appetite and inhibiting body fat biosynthesis. However, the exact mechanism of action of this novel phytopharmaceutical has yet to be fully understood. In a previous study, we showed that in the rat brain cortex a novel HCA extract (HCA-SX, Super CitriMax™) increases the release/availability of radiolabeled 5-hydroxytryptamine or serotonin ([3H]-5-HT), a neurotransmitter implicated in the regulation of eating behavior and appetite control. The aim of the present study was 2-fold: (a) to determine the effect of HCA-SX on 5-HT uptake in rat brain cortex in vitro; and (b) to evaluate the safety of HCA-SX in vivo. Isolated rat brain cortex slices were incubated in oxygenated Krebs solution for 20 min and transferred to buffer solutions containing [3H]-5-HT for different time intervals. In some experiments, tissues were exposed to HCA-SX (10 μM – 1 mM) and the serotonin receptor reuptake inhibitors (SRRI) fluoxetine (100 μM) plus clomipramine (10 μM). Uptake of [3H]-5-HT was expressed as d.p.m./mg wet weight. A time-dependent uptake of [3H]-5-HT occurred in cortical slices reaching a maximum at 60 min. HCA-SX, and fluoxetine plus clomipramine inhibited the time-dependent uptake of [3H]-5-HT. At 90 min, HCA-SX (300 μM) caused a 20% decrease, whereas fluoxetine plus clomipramine inhibited [3H]-5-HT uptake by 30%. In safety studies, acute oral toxicity, acute dermal toxicity, primary dermal irritation and primary eye irritation, were conducted in animals using various doses of HCA-SX. Results indicate that the LD50 of HCA-SX is greater than 5000 mg/kg when administered once orally via gastric intubation to fasted male and female Albino rats. No gross toxicological findings were observed under the experimental conditions. Taken together, these in vivo toxicological studies demonstrate that HCA-SX is a safe, natural supplement under the conditions it was tested. Furthermore, HCA-SX can inhibit [3H]-5-HT uptake (and also increase 5-HT availability) in isolated rat brain cortical slices in a manner similar to that of SRRIs, and thus may prove beneficial in controlling appetite, as well as treatment of depression, insomnia, migraine headaches and other serotonin-deficient conditions.

Role of hydrogen sulfide production in inhibitory action of L-cysteine on isolated porcine irides.

Purpose: To investigate the direct pharmacological actions of L-cysteine, a substrate for the production of H2S, on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we examined the underlying mechanism of action of L-cysteine in this smooth muscle. Methods: Isolated porcine iris muscle strips were set up in organ baths containing oxygenated Krebs buffer solution at 37°C. Longitudinal isometric tension was recorded via a grass FT03 Force-Displacement Transducer and analyzed using the PolyView computer software. The relaxant action of L-cysteine on carbachol-induced tone was studied in the absence and presence of inhibitors of enzymes of the biosynthetic pathways for H2S, and prostanoids. In addition, we also examined the effect of ATP-sensitive K+ (KATP) channel antagonist, glibenclamide on relaxations induced by L-cysteine. Results: L-cysteine (30 nM–1 mM) evoked concentration-dependent relaxations of carbachol-induced tone in isolated porcine irides, reaching a maximum inhibition of 43% at 1 mM. This response was enhanced significantly (P < 0.001) in the presence of the COX inhibitor, flurbiprofen (3 μM). Additionally,in the presence of flurbiprofen, the H2S donors, NaHS and Na2S, mimicked the relaxations produced by L-cysteine, yielding IC50 values of 5.8 μM and 180 μM, espectively. Both the inhibitor of cystathionine β-synthase, AOA (30 μM) and the KATP channel antagonist, glibenclamide (100 μM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to L-cysteine and attenuated the maximum inhibitory response. Conversely, the inhibitor of cystathionine γ-lyase, PAG (1 mM) blocked only relaxations caused by high concentrations of L-cysteine (> 100 μM). Conclusions: The inhibitory action of L-cysteine in isolated porcine irides is dependent on the endogenous production of H2S by cystathionine γ-lyase and cystathionine β-synthase. Furthermore, prostanoids and KATP channels are involved in the inhibitory action of L-cysteine in this tissue.

Hypoxia-induced [3H]D-aspartate release from isolated bovine retina: modulation by calcium-channel blockers and glutamatergic agonists and antagonists

Purpose. The aim of the present study was two-fold: (a) to examine the effect of hypoxia on [ 3 H]D-aspartate release from isolated bovine and human retinae, and (b) to investigate the regulation of hypoxia-induced neurotransmitter release by glutamate receptor agonists and antagonists. Methods. Isolated neural retinae were incubated in oxygenated Krebs buffer solution containing [ 3 H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [ 3 H]D-aspartate was evoked by K + (50 mM) applied at 90 minutes (S 1) and hypoxia (induced by exposure of tissues to solutions pregassed with 95%N 2 : 5% CO 2 for 60 minutes) at 108 minutes (S 2) after onset of superfusion. Results. Under hypoxic conditions, pO 2 in normal Krebs buffer solution was reduced from 14.53 ± 0.26 ppm (n = 6) to 0.54 ± 0.04 ppm (n = 9) after one hour of gassing with 95% N 2 : 5% CO 2. Exposure to hypoxia elicited an overflow of [ 3 H]D-aspartate yielding S 2 /S 1 ratios of 0.62 ± 0.06 (n = 12) and 0.54 ± 0.03 (n = 8) in bovine and human tissues respectively. In isolated bovine retinae, L- and N-calcium-channel antagonists diltiazem, nitrendipine, verapamil and ?-conotoxin significantly (p < 0.01 or higher) attenuated hypoxia-induced [ 3 H]D-aspartate release. L-glutamate (30 µM) significantly (p < 0.001) potentiated hypoxia-induced [ 3 H]D-aspartate release whereas kainate (30 µM) inhibited this response. NMDA (in concentrations up to 1 mM) had no effect on hypoxia-induced [ 3 H]D-aspartate release. Antagonists of glutamate receptors and the polyamine site on the NMDA receptor inhibited hypoxia-induced release of [ 3 H]D-aspartate in bovine retina with the following rank order of activity: ifenprodil ? MCPG > L-AP3 > MK-801. At an equimolar concentration (10 µM), L-AP3 but not ifenprodil, MCPG, MK 801 or arcaine, caused a significant (p < 0.001) inhibition of hypoxia-induced [ 3 H]D-aspartate release from human retinae. Conclusions. Hypoxia can induce the release of [ 3 H]D-aspartate from isolated bovine retinae by a calcium-dependent process. Hypoxia-induced [ 3 H]D-aspartate release from isolated bovine retinae can be regulated by glutamate receptor agonists/antagonists and blockers of polyamine site on the NMDA receptor.

Pharmacology of Serotonin Receptors Modulating Electrically-induced [3H]-Norepinephrine Release From Isolated Mammalian Iris-Ciliary Bodies

The pharmacology of prejunctional serotonin (5-HT) heteroreceptors that regulate the release of norepinephrine (NE) was studied in isolated bovine and human iris-ciliary bodies. The effect of exogenous 5-HT and various 5-HT receptor agonists was examined on the release of [3H]-norepinephrine ([3H]NE). Both 5-HT and m-chlorophenyl-biguanide (m-CPBG) caused enhancement in the field-stimulated release of [3H]NE from bovine tissues whereas 5-carboxamidotryptamine (5-CT) had no such effect. On the other hand, 8hydroxy-dipropylaminotetralin (8-OH-DPAT), caused a significant dose-related inhibition of evoked [3H]NE release. In human iris-ciliary bodies, 5-HT caused an inhibitory response on electrically-evoked [3H]NE release at low concentrations but produced an excitatory action at concentrations greater than 3 microM. To further confirm the nature of the prejunctional 5-HT heteroreceptors regulating [3H]NE release, effects of 5-HT3, 5-HT6 and 5-HT7 receptor antagonists were examined on a standard response to 5-HT. All antagonists examined caused a concentration-dependent inhibition of the response elicited by the standard 5-HT-induced response with the following rank order of potency (as measured by IC30 values): MDL-72222 >> SB-258719 > RO-04-690. We conclude that the excitatory prejunctional 5-HT heteroreceptors present in bovine iris-ciliary bodies belong to the 5-HT3 receptor subtype.

Potentiation of sympathetic neurotransmission in bovine isolated irides by isoprostanes

Isoprostanes (IsoP) are formed by free radical catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. In the present study, we examined the effect of IsoP on norepinephrine (NE) release from the bovine isolated iris. Furthermore, we studied the role of IsoPs in hydrogen peroxide (H2O2)-induced enhancement of NE release from this tissue. Isolated bovine irides were prepared for studies of [3H]NE release using the superfusion method. Release of [3H]NE was induced via electrical field stimulation. Both 8-iso-prostaglandin E2 (E2-IsoP) and 8-iso-prostaglandin F2α (F2-IsoP) produced a concentration-related enhancement of field-stimulated [3H]NE release from isolated bovine irides, an effect that was mimicked by the thromboxane (Tx) receptor agonist, U46619 and by H2O2. The Tx-receptor antagonist, SQ 29548 inhibited responses to E2-IsoP (10μM) with an IC50 of 370±50 nM. SQ 29548 (10μM) also blocked the enhancement of electrically-evoked [3H]NE release induced by U46619 (10μM) but not that caused by H2O2 (300μM). The Tx synthetase inhibitor, carboxyheptylimidazole (10μM) prevented the stimulatory effect of E2-IsoP on evoked [3H]NE release without affecting responses induced by H2O2. We conclude that IsoPs can enhance sympathetic neurotransmission in the bovine isolated iris, an effect that can be blocked by a Tx-receptor antagonist. Furthermore, endogenously produced Txs mediate the stimulatory effect of IsoPs on NE release. However, endogenously generated IsoPs or Txs are not involved in H2O2-induced potentiation of sympathetic neurotransmission.

Arachidonic acid metabolites and peroxide-induced inhibition of [ 3H]D-aspartate release from bovine isolated retinae

We have previously shown that hydrogen peroxide (H 2 O 2) can inhibit K + -depolarization-evoked [ 3 H]D-aspartate release from bovine isolated retinae. In the present study, we investigated the role of arachidonic acid metabolites in the inhibitory response elicited by H 2 O 2 in the bovine retinae. Furthermore, we examined the direct effect of H 2 O 2 on the production of prostaglandins and isoprostanes in this tissue. Isolated bovine retinae were prepared for studies of [ 3 H]D-aspartate release using the Superfusion Method. Release of [ 3 H]D-aspartate was elicited by Krebs solution containing an iso-osmotic concentration of KCl (50mM). A direct action of H 2 O 2 on prostaglandin E 2 (PGE2) and 8-isoprostane F 2a (8-iso-PGF 2a) was also measured by enzyme-linked immunosorbant assay (ELISA). The cyclooxygenase inhibitor, flurbiprofen (3µM), or the thromboxane-receptor antagonist, SQ 29548 (10µM) had no significant (p > 0.05) effect on K + -evoked [ 3 H]D-aspartate release. On the other hand, both flurbiprofen (3µM) and SQ 29548 (10µM) blocked the inhibition of K + -evoked [ 3 H]D-aspartate induced by H 2 O 2 (30µM). In concentrations up to 100µM, H 2 O 2 caused an increase in PGE 2 and 8-iso-PGF 2a over basal levels. For instance, H 2 O 2 (100µM) increased PGE 2 and 8-iso-PGF 2a over basal levels by 348 ± 41% and 185 ± 26 (n = 4), respectively. We conclude that the peroxide-mediated inhibition of [ 3 H]D-aspartate may involve the production of prostaglandins and isoprostanes in the bovine isolated retinae.

Effect of inhibition of cyclooxygenase on pre- and postjunctional actions of peroxides in the iris-ciliary body.

In the present study, we investigated the effect of inhibition of cyclooxygenase (COX) with flurbiprofen (FBF) on peroxide-induced enhancement of field-stimulated [3H]norepinephrine ([3H]NE) release from bovine isolated irides. Furthermore, the effect of FBF was examined on peroxide-induced attenuation of contractions evoked by carbachol on this tissue. Irides were prepared for studies of neurotransmitter release and for measurement of contractile tension in vitro. Pretreatment of tissues with FBF (10 μM) caused significant (P < 0.001) rightward shifts of concentration-response curves to H2O2 and also decreased cumene hydroperoxide (cuOOH)-induced enhancement of evoked [3H]NE release. FBF (10 μM) partially prevented the attenuation of carbachol-induced contractions induced by H2O2 (300 μM) and cuOOH (300 μM). We conclude that inhibition of the biosynthesis of prostanoids reduced both the prejunctional stimulatory effects of H2O2 and cuOOH on sympathetic neurotransmission and inhibitory effects of peroxides on carbachol-induced contractions the in the bovine isolated iris.

Glucose-Deprivation-Induced [3H]D-Aspartate Release from Isolated Bovine and Human Retinae

The glucose deprivation-induced release of [3H]D-aspartate was studied in bovine and human retinas in a superfusion apparatus. [3H]D-aspartate release was significantly increased upon omitting glucose in the superfusion buffer. This effect was dependent on external Ca2+ because L- and N-type Ca2+-channel blockers, such as diltiazem (1 microM), nitrendipine (1 microM), and omega-conotoxin (100 nM), significantly reduced the effect of glucose-deprivation induced release of [3H]D-aspartate. Furthermore, while glutamate receptor agonists (L-glutamate, N-methyl-D-aspartate, but not kainate) potentiated the effects of glucose deprivation, antagonists (MK-801, MCPG, ifenprodil, and L-AP3) at these receptors blocked the glucose deprivation-induced release process. Taken together, these studies have demonstrated that under conditions of glucose deprivation, as may happen during ischemic events in vivo, the retinal glutamatergic nerve endings and/or glial cells promote the efflux of [3H]D-aspartate into the extracellular environment. This process appears to be receptor-mediated and dependent on extracellular Ca2+ and is similar to previous reports pertaining to brain tissues.