Sze Wa Chan

Separation of emetic and anorexic responses of exendin-4, a GLP-1 receptor agonist in Suncus murinus (house musk shrew).

The use of glucagon-like peptide-1 (7-36) amide (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus is commonly associated with nausea and vomiting. Therefore, the present studies investigated the potential of GLP-1 receptor ligands to modulate emesis and feeding in Suncus murinus. Exendin-4, a selective GLP-1 receptor agonist, was administered subcutaneously (1-30 nmol/kg) or intracerebroventricularly (0.03-3 nmol) after 12-h of fasting. In other studies, animals were pretreated with the GLP-1 receptor antagonist, exendin (9-39), or saline (5 μl) 15 min prior to exendin-4 (3 nmol, i.c.v.). Behaviour of animals and food and water intake were then recorded for 1-2 h; c-Fos expression was also assessed in the brains of animals in the i.c.v. studies. The subcutaneous administration of exendin-4 reduced food and water intake (p < 0.001) and induced emesis in 40% of animals (p > 0.05). The intracerebroventricular administration of exendin-4 also prevented feeding, and induced emesis (p < 0.01). In these studies, exendin (9-39) (30 nmol, i.c.v.) antagonised emesis induced by exendin-4 and the increased c-Fos expressions in the brainstem and hypothalamus (p < 0.05), but it was ineffective in reversing the exendin-4-induced inhibition of food and water intake (p > 0.05). These data suggest that exendin-4 exerts its emetic effects in the brainstem and/or hypothalamus via GLP-1 receptors. The action of exendin-4 to suppress feeding may involve non-classical GLP-1 receptors or other mechanisms.

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection.

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 degrees C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and gamma-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.

Modulation by homocysteine of the iberiotoxin-sensitive, Ca2+-activated K+ channels of porcine coronary artery smooth muscle cells

We evaluated the acute effect of homocysteine on the iberiotoxin-sensitive, Ca(2+)-activated K(+) (BK(Ca)) channels of the porcine coronary artery smooth muscle cells. NS 1619 (1 to 30 microM) caused a concentration-dependent enhancement of the BK(Ca) amplitude (recorded using the whole-cell, membrane-rupture configuration) only with an elevated [Ca(2+)](i) of approximately 444 nM, but not with [Ca(2+)](i) of approximately 100 nM. Homocysteine (30 microM) caused a small inhibition ( approximately 16%) of the BK(Ca) amplitude ([Ca(2+)](i)= approximately 444 nM), and a greater inhibition ( approximately 77%) was observed with 100 microM NADH present in the pipette solution. The inhibition persisted after washing. With NADPH (100 microM), a smaller magnitude of inhibition ( approximately 34%) of the BK(Ca) amplitude was recorded. The NS 1619-mediated enhancement of the BK(Ca) amplitude (with elevated [Ca(2+)](i) plus NADH in the pipette) was attenuated by homocysteine. The homocysteine-mediated inhibition of the BK(Ca) amplitude was suppressed by Tiron (10 mM) or diphenylene iodonium (30 nM), applied alone, but not by superoxide dismutase (500 U/ml) and catalase (500 U/ml). Generation of superoxide (O(2)(-)) of the smooth muscle cells (with NADH presence), measured using the lucigenin-enhanced chemiluminescence, was markedly increased by angiotensin II (100 nM) and homocysteine (30 microM). The chemiluminescence signal was sensitive to apocynin (300 microM) or Tiron, applied alone, but not to superoxide dismutase and catalase. In conclusion, our results demonstrate that acute homocysteine application inhibits the iberiotoxin-sensitive BK(Ca) channels (with elevated [Ca(2+)](i) and NADH present) which is probably caused by the NADH oxidase activation and the concomitant generation of intracellular superoxide.

A physiological role of glucagon-like peptide-1 receptors in the central nervous system of Suncus murinus (house musk shrew)

Glucagon-like peptide-1 (7-36) amide (GLP-1) is released from the gut as an incretin hormone to stimulate glucose-stimulated insulin secretion. GLP-1 is also produced in the central nervous system (CNS) as a neurotransmitter that regulates feeding behaviour. By using polyclonal antiserum against GLP-1 and GLP-1 receptors, we identified the distribution of GLP-1 immunoreactive fibres and GLP-1 receptor immunoreactivity in the ventromedial hypothalamus of Suncus murinus (house musk shrew). In functional studies, subcutaneous administration of exendin-4 (1 - 30 nmol/kg) reduced blood glucose levels dose-dependently by up to 49% during an intraperitoneal glucose tolerance test (P<0.001). The glucose-lowering effects were also observed after an intracerebroventricular (i.c.v.; 0.3 - 3 nmol) or intracerebral ventromedial hypothalamic microinfusion (iVMH; 0.3 - 3 pmol) of exendin-4. The area under the curve values for glucose after i.c.v. and iVMH administrations of exendin-4 were reduced by up to 53% (P<0.01) and 46% (P<0.01), respectively. Exendin-4 (i.c.v.; 3 nmol) also increased glucose-stimulated insulin secretion by 20% compared to controls (P<0.05). The GLP-1 receptor antagonist, exendin (9-39) (10 nmol, i.c.v.) did not modify blood glucose levels but it antagonized the glucose-lowering effect of exendin-4 (1 nmol, i.c.v.; P<0.05). The data suggests that the central GLP-1 system may regulate glucose homeostasis by increasing insulin secretion. Further, GLP-1 receptors in the ventromedial hypothalamus appear to play an important role in the regulation of glucose homeostasis in S. murinus.

Contractile effect of tachykinins on Suncus murinus (house musk shrew) isolated ileum

Recent studies used Suncus murinus to investigate the anti-emetic potential of NK(1) tachykinin receptor antagonists. However, the pharmacology of tachykinin receptors in this species has not been fully characterized. In the present studies, therefore, we examined a range of tachykinin receptor agonists for a capacity to induce contractions of the isolated ileum. The tachykinin NK1 receptor preferring agonists substance P, septide and [Sar9Met(O2)11] substance P, and the tachykinin NK2 preferring agonists neurokinin A and GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) caused concentration dependent contractions with EC50 values in the nanomolar range. However, the tachykinin NK3 preferring agonists neurokinin B and senktide (1nM-1microM) induced only weak contractions. The action of senktide, but not [Sar9Met(O2)11] substance P, septide, or GR 64349, was antagonized significantly by atropine (P<0.05); tetrodotoxin and hexamethonium were inactive. The tachykinin NK1 receptor antagonist CP-99,994 ((+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine]) (10-100nM) inhibited substance P- and septide-induced contractions non-competitively. The pA2 value estimated for CP-99,994 against septide was 7.3+/-0.1. It also non-competitively antagonized the contractile responses induced by [Sar9Met(O2)11] substance P with a pA2 of 7.4+/-0.1. CP-99,994 also had a slight inhibitory action on neurokinin A-induced contractions, but did not modify the action of GR 64349. Conversely, the tachykinin NK2 receptor antagonist, saredutant, competitively antagonized GR 64349-induced contractions with a pA2 of 7.34+/-0.02. On the other hand, the presence of both CP-99,994 and saredutant competitively antagonized substance P-induced contraction. The present studies indicate that tachykininNK1 and NK2 receptors exist in the ileum of S. murinus and are involved in mediating contractions directly on smooth muscle, whereas tachykinin NK3 receptors may play a minor role involving a release of acetylcholine.

Differential hypoglycaemic, anorectic, autonomic and emetic effects of the glucagon-like peptide receptor agonist, exendin-4, in the conscious telemetered ferret

BackgroundRodents are incapable of emesis and consequently the emetic potential of glucagon-like peptide-1 receptor (GLP-1R) agonists in studies designed to assess a potential blood glucose lowering action of the compound was missed. Therefore, we investigated if the ferret, a carnivore with demonstrated translation capability in emesis research, would identify the emetic potential of the GLP-1R agonist, exendin-4, and any associated effects on gastric motor function, appetite and cardiovascular homeostasis.MethodsThe biological activity of the GLP-1R ligands was investigated in vivo using a glucose tolerance test in pentobarbitone-anesthetised ferrets and in vitro using organ bath studies. Radiotelemetry was used to investigate the effect of exendin-4 on gastric myoelectric activity (GMA) and cardiovascular function in conscious ferrets; behaviour was also simultaneously assessed. Western blot was used to characterize GLP-1R distribution in the gastrointestinal and brain tissues.ResultsIn anesthetised ferrets, exendin-4 (30 nmol/kg, s.c.) reduced experimentally elevated blood glucose levels by 36.3%, whereas the GLP-1R antagonist, exendin (9–39) (300 nmol/kg, s.c.) antagonised the effect and increased AUC0–120 by 31.0% when injected alone (P < 0.05). In animals with radiotelemetry devices, exendin-4 (100 nmol/kg, s.c.) induced emesis in 1/9 ferrets, but inhibited food intake and decreased heart rate variability (HRV) in all animals (P < 0.05). In the animals not exhibiting emesis, there was no effect on GMA, mean arterial blood pressure, heart rate, or core body temperature. In the ferret exhibiting emesis, there was a shift in the GMA towards bradygastria with a decrease in power, and a concomitant decrease in HRV. Western blot revealed GLP-1R throughout the gastrointestinal tract but exendin-4 (up to 300 nM) and exendin (9–39), failed to contract or relax isolated ferret gut tissues. GLP-1R were found in all major brain regions and the levels were comparable those in the vagus nerve.ConclusionsPeripherally administered exendin-4 reduced blood glucose and inhibited feeding with a low emetic potential similar to that in humans (11% vs 12.8%). A disrupted GMA only occurred in the animal exhibiting emesis raising the possibility that disruption of the GMA may influence the probability of emesis occurring in response to treatment with GLP-1R agonists.