Amperometric measurement of glutamate release modulation by gabapentin and pregabalin in rat neocortical slices: role of voltage-sensitive Ca2+ α2δ-1 subunit

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.     

Bradykinin activates a cross-signaling pathway between sensory and adrenergic nerve endings in the heart: a novel mechanism of ischemic norepinephrine release?

We had shown that bradykinin (BK) generated by cardiac sympathetic nerve endings (i.e., synaptosomes) promotes exocytotic norepinephrine (NE) release in an autocrine mode. Because the synaptosomal preparation may include sensory C-fiber endings, which BK is known to stimulate, sensory nerves could contribute to the proadrenergic effects of BK in the heart. We report that BK is a potent releaser of NE from guinea pig heart synaptosomes (EC(50) approximately 20 nM), an effect mediated by B(2) receptors, and almost completely abolished by prior C-fiber destruction or blockade of calcitonin gene-related peptide and neurokinin-1 receptors. C-fiber destruction also greatly decreased BK-induced NE release from the intact heart, whereas tyramine-induced NE release was unaffected. Furthermore, C-fiber stimulation with capsaicin and activation of calcitonin gene-related peptide and neurokinin-1 receptors initiated NE release from cardiac synaptosomes, indicating that stimulation of sensory neurons in turn activates sympathetic nerve terminals. Thus, BK is likely to release NE in the heart in part by first liberating calcitonin gene-related peptide and Substance P from sensory nerve endings; these neuropeptides then stimulate specific receptors on sympathetic terminals. This action of BK is positively modulated by cyclooxygenase products, attenuated by activation of histamine H(3) receptors, and potentiated at a lower pH. The NE-releasing action of BK is likely to be enhanced in myocardial ischemia, when protons accumulate, C fibers become activated, and the production of prostaglandins and BK increases. Because NE is a major arrhythmogenic agent, the activation of this interneuronal signaling system between sensory and adrenergic neurons may contribute to ischemic dysrhythmias and sudden cardiac death.     

Effect of β-sodium aescinate on hypoxia-inducible factor-1α expression in rat brain cortex after cardiopulmonary resuscitation

BACKGROUND:

This study was undertaken to investigate the expression of hypoxia-inducible factor-1α (HIF-1α) in rat cerebral cortex and the effects of β-sodium aescinate (SA) administration after return of spontaneous circulation (ROSC).

METHODS:

Sixty rats were divided into three groups: SA group, injected intraperitoneally with SA instantly after ROSC; control group, injected intraperitoneally with normal saline; and sham-operated group, without cardiac arrest or SA. The cardiac arrest model was established using asphyxiation and intravenous potassium chloride. Blood was sampled 1, 6, 12, and 24 hours after ROSC. Protein and mRNA levels of HIF-1α, VEGF and EPO were detected in the cerebral cortex by immunohistochemistry and real-time RT-PCR; serum levels of NSE and S100β were determined by enzyme-linked immunosorbent assays.

RESULTS:

Serum S100β and NSE were significantly increased in the control group versus the sham-operated group 1, 6, 12 and 24 hours after ROSC (P<0.05). Protein and mRNA levels of HIF-1α, VEGF and EPO were significantly increased in the control rats (P<0.05). Serum NSE and S100β were significantly decreased in the SA group versus the control group 1, 6, 12 and 24 hours after ROSC (P<0.05). Protein and mRNA levels of HIF-1α, VEGF and EPO were significantly increased in the SA group (P<0.05).

CONCLUSIONS:

The expression of HIF-1α is increased in rat cerebral cortex after ROSC, and SA up-regulates the expression of HIF-1α. The up-regulation of HIF-1α improves the resistance of the cortex to ischemia and hypoxia and contributes to neuroprotection, possibly because of up-regulation of EPO and VEGF expression.KEY WORDS: Cardiopulmonary resuscitation, HIF-1α, Erythropoietin, Vascular endothelial growth factor, β-sodium aescinate, Neuroprotection     

Modulation of neuronal activity in the nucleus raphé magnus by the 5-HT(1)-receptor agonist naratriptan in rat

Modulatory effects of the new antimigraine drug naratriptan, a 5-HT₁-receptor-agonist, on neurons of the nucleus raphé magnus were examined in rat by extracellular recordings. In the nucleus raphé magnus neuronal activity decreased in on-cells and increased in off-cells after intravenous administration of naratriptan. The modulatory effects of naratriptan were similar to the well-known effects of morphine on neurons in the nucleus raphé magnus. The results of this study suggest central actions of naratriptan and may point to an involvement of the endogenous pain control system in the antinociceptive effects of the 5-HT₁-receptor-agonist.     

(1→3)-β-d-glucan determination in rat organs with limulus coagulation factor G

One cause of a false positive limulus test after surgery or hemodialysis has been identified as extrinsic (1→3)-β-d-glucan which was derived from surgical gauze or cellulose dialyzer. However, there have been no investigations concerning intrinsic factors and the presence of (1→3)-β-d-glucan in mammalian organs. In this study, (1→3)-β-d-glucan in homogenate of various rat organs and stool was measured by a Gluspecy test (G test) using factor G, which specifically reacts with (1→3)-β-d-glucan. In small intestine and lung, large amounts of factor G-activating substance were observed and identified as (1→3)-β-d-glucan by a digestion study using end-(1→3)-β-d-glucanase. However, only very small amounts of (1→3)-β-d-glucan were found in the kidney, spleen, vena cava, aorta, thymus, heart and liver. In serum and plasma, no (1→3)-β-d-glucan was observed. On the other hand, extremely large amounts of (1→3)-β-d-glucan were found in stool. Minute amounts of (1→3)-β-d-glucan were observed in a variety organs except for the small intestine and lung. High levels of (1→3)-β-d-glucan found in the small intestine might be traced to contamination by stool in the small intestine, and such levels in the lung might derive from macrophages which have trapped (1→3)-β-d-glucan in the air.     

The relativity of the activity change of tissue—type plasminogen activator and type 1 plasminogen activator inhibitor in patients with cerebral infarction and defect of nerve function

AIM：To explore the dynamic changes of the activity of tissue-type plasminogen activator(t-PA) and type 1 plasminogen activator inhibitor (PAI-1) and its clinical significance by observing the activity of t-PA and PAI-1 in patients in acute and recovery phases of arteriosclerotic cerebral infarction.METHODS:Testing the activity of plasme t-PA and PAI-1 of 91 patients with arteriosclerotic cerebral infarction and 80 healthy old ages by Chromgenic substrate methods and controlling them.RESULTS:The activity of t-PA in acute and recovery stage of arteriosclerotic cerebral infarction patients were apparently lower than that of control and the activity of PAI-1 were higher than that of control.volume of cerebral infarction was negatively related to the activity of t-PA and positively related to the activity of PAI-1.CONCLUSION:The plasma fibrinolytic activities of the acute and recovery stage patients with arteriosclerotic cerebral infarction declined.     

Differential upregulation in DRG neurons of an α2δ-1 splice variant with a lower affinity for gabapentin after peripheral sensory nerve injury

The α₂δ-1 protein is an auxiliary subunit of voltage-gated calcium channels, critical for neurotransmitter release. It is upregulated in dorsal root ganglion (DRG) neurons following sensory nerve injury, and is also the therapeutic target of the gabapentinoid drugs, which are efficacious in both experimental and human neuropathic pain conditions. α₂δ-1 has 3 spliced regions: A, B, and C. A and C are cassette exons, whereas B is introduced via an alternative 3′ splice acceptor site. Here we have examined the presence of α₂δ-1 splice variants in DRG neurons, and have found that although the main α₂δ-1 splice variant in DRG is the same as that in brain (α₂δ-1 ΔA+B+C), there is also another α₂δ-1 splice variant (ΔA+BΔC), which is expressed in DRG neurons and is differentially upregulated compared to the main DRG splice variant α₂δ-1 ΔA+B+C following spinal nerve ligation. Furthermore, this differential upregulation occurs preferentially in a small nonmyelinated DRG neuron fraction, obtained by density gradient separation. The α₂δ-1 ΔA+BΔC splice variant supports Ca_V2 calcium currents with unaltered properties compared to α₂δ-1 ΔA+B+C, but shows a significantly reduced affinity for gabapentin. This variant could therefore play a role in determining the efficacy of gabapentin in neuropathic pain.     

Cannabinoid Receptor Activation Correlates with the Proapoptotic Action of the β2-Adrenergic Agonist (R,R')-4-Methoxy-1-Naphthylfenoterol in HepG2 Hepatocarcinoma Cells

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with β(2)-adrenergic receptor (β(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the β(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of β(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a β(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of β(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The β(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting β(2)-AR-CBR ligand.