Blockade Of Voltage-dependent Calcium Channels Research Articles

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Spasmolytic effect of Acmella oleracea flowers extract on isolated rat ileum

Introduction: Acmella oleracea has been used as a traditional medicine for the treatment of asthma, sore throat, haemorrhoids and toothache. However, whether A. oleracea has gastrointestinal functions, such as regulation of intestinal contractions, has not been fully elucidated. Therefore, the aim of the present study was to investigate the effect of A. oleracea flowers extract (AFE) on rat ileum contractions and the possible mechanism(s) of its action. Methods: The extract was prepared using the Soxhlet apparatus with 95% ethanol. Ileum was removed from male Wistar rats and mounted in an organ bath containing Krebs solution. The tissue contractions were recorded by an isotonic transducer under 1 g tension.Results: The cumulative concentrations of the AFE (0.01–1 mg/mL) reduced the ileum contractions induced by KCl (80 mM) (n = 6, P < 0.05). AFE (1 mg/mL) attenuated the contractions induced by cumulative concentrations of CaCl2 (1–20 mM), while the spasmolytic effects of the extract were not reduced after tissue incubation with N (ω)-nitro-L-arginine methyl ester (L-NAME) (100 μM, 20 minutes).Conclusion: These results suggest that AFE inhibits ileum contractions without involving the nitric oxide pathway, which is possibly mediated via blockade of voltage-dependent calcium channels. A. oleracea may be useful in gastrointestinal disorders such as diarrhoea.

Hydroxyl Group and Vasorelaxant Effects of Perillyl Alcohol, Carveol, Limonene on Aorta Smooth Muscle of Rats.

The present study used isometric tension recording to investigate the vasorelaxant effect of limonene (LM), carveol (CV), and perillyl alcohol (POH) on contractility parameters of the rat aorta, focusing in particular on the structure-activity relationship. LM, CV, and POH showed a reversible inhibitory effect on the contraction induced by electromechanical and pharmacomechanical coupling. In the case of LM, but not CV and POH, this effect was influenced by preservation of the endothelium. POH and CV but not LM exhibited greater pharmacological potency on BayK-8644-induced contraction and on electromechanical coupling than on pharmacomechanical coupling. In endothelium-denuded preparations, the order of pharmacological potency on electrochemical coupling was LM < CV < POH. These compounds inhibited also, with grossly similar pharmacological potency, the contraction induced by phorbol ester dibutyrate. The present results suggest that LM, CV and POH induced relaxant effect on vascular smooth muscle by means of different mechanisms likely to include inhibition of PKC and IP3 pathway. For CV and POH, hydroxylated compounds, it was in electromechanical coupling that the greater pharmacological potency was observed, thus suggesting a relative specificity for a mechanism likely to be important in electromechanical coupling, for example, blockade of voltage-dependent calcium channel.

The vasorelaxant effect of Canarium odontophyllum Miq. (Dabai) extract in rat thoracic aorta

Background/aimCanarium odontophyllum (CO) (Miq.) (Burseraceae) has been traditionally consumed for its antioxidant benefit in maintaining longetivity. However, there is no report concerning its pharmacological activity on blood vessels. This study was aimed to investigate the effects of CO aqueous leaf extract on isolated aortic rings. MethodsIsolated thoracic aortic rings were suspended in a tissue bath and placed between 2 tungsten wires and connected to an isometric force transducer. The tension changes were recorded to a data acquisition system. ResultsThe leaf aqueous extract (3–15 mg/ml) induced relaxation in endothelium-intact and endothelium-denuded aortic rings precontracted with PE. Incubation with nitric oxide enzyme inhibitor L-NAME, however, did not alter the CO-induced vasorelaxant on endothelium-intact aortic ring. The vasorelaxant effect of CO was endothelium-independent as it reduced the contraction induced by extracellular Ca2+ in endothelium-denuded aortic ring precontracted by PE and KCl in Ca2+-free Krebs solution. However, CO did not inhibit the release of intracellular Ca2+ from sarcoplasmic reticulum. ConclusionThis study suggests that the vasorelaxant effect of aqueous extract from CO leaves was endothelium-independent and was possibly mediated through the blockade of voltage-dependent calcium channel and receptor-operated calcium channel.

Nongenomic bronchodilating action elicited by dehydroepiandrosterone (DHEA) in a guinea pig asthma model

Primates secrete large amounts of the precursor steroid dehydroepiandrosterone (DHEA); in humans, its levels are low during childhood and start declining after the fourth decade. It has been postulated that the progressive decline in DHEA levels may be related with the severity of asthma associated with age. To determine whether DHEA may regulate the airway smooth muscle (ASM) activity, isolated tracheal rings with and without epithelium from male guinea pigs were isometrically recorded to characterize the response of ASM to DHEA at different concentrations on KCl- and carbachol (CCh)-induced contraction as well as on ovalbumin (OVA)-induced contraction in sensitized guinea pigs. Additionally, we used barometric plethysmography in sensitized guinea pigs in order to compare changes of the lung resistance increased by the antigen challenge to OVA in the absence and presence of different doses of DHEA. DHEA concentration-dependently abolished the contraction to KCl, CCh and OVA, and no differences were found in preparations with and without epithelium. DHEA-induced relaxation was not modified by the suppression of protein synthesis or transcription, pharmacological inhibition of nitric oxide (NO) synthase, nor by antagonist of β2-adrenergic receptors or an inhibitor of the 3β-HSD enzyme. Likewise, Ca2+-induced contraction in Ca2+-free depolarized tissues was antagonized by DHEA, and the contraction to the L-type voltage-dependent calcium channel activator (Bay K 8644) was inhibited by DHEA. Furthermore, DHEA prevented OVA-induced increases in lung resistance. These results indicate that DHEA-induced relaxation in ASM is a nongenomic (membrane) action and is not produced after its bioconversion. The data suggest that DHEA-induced relaxation is an epithelium- and NO-independent mechanism that involves a blockade of voltage-dependent calcium channels and possible non-selective cation channels.

Leptin Enhances Nitric Oxide-Dependent Relaxation of the Clitoral Corpus Cavernosum

PurposeThe effects of leptin on female sexual behaviors are controversial, and studies on this topic are limited. The objectives of this study were to evaluate the direct effects of leptin on clitoral vasoreactivity in vitro and to determine the mechanism of action.Materials and MethodsIsometric tension studies were conducted to determine the effects of pretreatment with leptin (10-8 M) on the contractile responses of rabbit clitoral corpus cavernosal smooth muscle strips. The effects of leptin were assessed on precontraction induced by phenylephrine (PE; 10-9-10-4 M) and KCl (35-140 mM). We also examined the effect of leptin on relaxation induced by acetylcholine (ACh; 10-9-10-4 M), verapamil (10-10-10-6 M), and sodium nitroprusside (10-9-10-4 M) in PE-precontracted (10-5 M) strips.ResultsLeptin enhanced ACh-induced relaxation in PE-precontracted strips. L-NAME pretreatment significantly reduced the effect of leptin on ACh-induced relaxation, whereas L-arginine potentiated the effect of leptin. Leptin decreased the KCl-induced contractile responses. Leptin increased verapamil-induced relaxation responses. The relaxation effects of leptin on KCl-induced contraction were inhibited by 10-5 M methylene blue and L-NAME pretreatment.ConclusionsA high concentration of leptin enhances ACh-dependent relaxation in clitoral cavernosal smooth muscles. These relaxation effects of leptin may occur through an NO-dependent mechanism and voltage-dependent calcium channel blockade.

Antispasmodic and vasodilator activities of Morinda citrifolia root extract are mediated through blockade of voltage dependent calcium channels

BackgroundMorinda citrifolia (Noni) is an edible plant with wide range of medicinal uses. It occurs exclusively in tropical climate zone from India through Southeast Asia and Australia to Eastern Polynesia and Hawaii. The objective of this study was to explore the possible mode(s) of action for its antispasmodic, vasodilator and cardio-suppressant effects to rationalize its medicinal use in gut and cardiovascular disorders.MethodsIsolated tissue preparations such as, rabbit jejunum, rat and rabbit aorta and guinea pig atria were used to test the antispasmodic and cardiovascular relaxant effects and the possible mode of action(s) of the 70% aqueous-ethanolic extract of Morinda citrifolia roots (Mc.Cr).ResultsThe Mc.Cr produced a concentration-dependent relaxation of spontaneous and high K+ induced contractions in isolated rabbit jejunum preparations. It also caused right ward shift in the concentration response curves of Ca++, similar to that of verapamil. In guinea-pig right atria, Mc.Cr caused inhibition of both atrial force and rate of spontaneous contractions. In rabbit thoracic aortic preparations, Mc.Cr also suppressed contractions induced by phenylephrine (1.0 μM) in normal- Ca++ and Ca++-free Kerb's solutions and by high K+, similar to that of verapamil. In rat thoracic aortic preparations, Mc.Cr also relaxed the phenylephrine (1.0 μM)-induced contractions. The vasodilatory responses were not altered in the presence of L-NAME (0.1 mM) or atropine (1.0 μM) and removal of endothelium.ConclusionsThese results suggest that the spasmolytic and vasodilator effects of Mc.Cr root extract are mediated possibly through blockade of voltage-dependent calcium channels and release of intracellular calcium, which may explain the medicinal use of Morinda citrifolia in diarrhea and hypertension. However, more detailed studies are required to assess the safety and efficacy of this plant.

Suppression of Formalin-Induced Nociception by Cilnidipine, a Voltage-Dependent Calcium Channel Blocker

Cilnidipine is a 1,4-dihydropyridine-derived voltage-dependent calcium channel (VDCC) blocker and suppresses N-type VDCC currents in addition to L-type VDCC currents. An earlier investigation has suggested that intrathecally injected cilnidipine produces antinociception by blocking N-type VDCCs in mice. The present study using the rat formalin model examined antinociceptive effects of intrathecally and orally administered cilnidipine to elucidate a putative site of antinociception of cilnidipine, assess the efficacy of oral cilnidipine for pain relief, and clarify the mechanism(s) responsible for the antinociceptive effect of oral cilnidipine. Cilnidipine (whether intrathecal or oral) suppressed nociception in phases 1 and 2 of the formalin model. In addition, the potency of oral cilnidipine to suppress formalin-induced nociception in phase 2 was greater than that of oral gabapentin, a clinically available drug for treatment of neuropathic pain. Cilnidipine elicited antinociceptive effects without neurological side-effects including serpentine-like tail movement, whole body shaking, and allodynia. Such side-effects can be induced by higher doses of intrathecal ziconotide, a clinically available N-type VDCC blocker. In contrast, orally administered nifedipine, an L-type VDCC blocker, had no effect on either phase of formalin-induced nociception. These results suggest that cilnidipine acts on the spinal cord to produce antinociception and is efficacious for pain relief after oral administration with better safety profile than that of ziconotide. Furthermore, the failure of orally administered nifedipine to affect formalin-induced nociception raises the possibility that oral cilnidipine produces antinociception through, at least in part, spinal N-type VDCC blockade.

New multipotent tetracyclic tacrines with neuroprotective activity

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues ( 5– 13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca 2+ overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor ( 11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of β-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70 mM K + and 60 μM H 2O 2. Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.

Calcium channels modulate nociceptive transmission in the trigeminal nucleus of the cat

Clinical observations and genetic studies have suggested a role for high-threshold voltage-dependent calcium channels (VDCCs) in the pathogenesis of migraine. This study investigated the role of P/Q-, L- and N-type VDCCs in post-synaptic action potential generation in trigeminovascular nociceptive afferents in the trigeminocervical complex (TCC) of the cat in vivo. Trigeminovascular nociceptive afferents were identified in the TCC by electrical stimulation of the superior sagittal sinus. Forty-six cell bodies were identified by their response to microiontophoresis of l-glutamate and their bipolar action potential shape. Blockade of VDCCs was accomplished by microiontophoresis of ω-agatoxin IVa/TK (P/Q-), ω-conotoxin GVIa (N-) and calciseptine (L-type). Non-selective antagonism was studied using cadmium ions. Non-selective blockade of high threshold VDCC with cadmium resulted in a reduction in l-glutamate-evoked neuronal activity ( P=0.01). Blockade of P/Q: TK- ( P<0.001), IVA- ( P=0.007), L- ( P<0.001) and N-type ( P<0.001) VDCCs resulted in significant reductions in post-synaptic action potential generation in response to l-glutamate. High threshold VDCCs, including P/Q-, L- and N-type VDCCs, can therefore modulate nociceptive transmission in the trigeminocervical complex in vivo. We discuss the evidence to suggest a role for VDCCs in the pathophysiology of primary headache disorders, and how abnormalities of function may contribute to their pathogenesis.

Ginger lowers blood pressure through blockade of voltage-dependent calcium channels.

Ginger (Zingiber officinale Roscoe), a well-known spice plant, has been used traditionally in a wide variety of ailments including hypertension. We report here the cardiovascular effects of ginger under controlled experimental conditions. The crude extract of ginger (Zo.Cr) induced a dose-dependent (0.3-3 mg/kg) fall in the arterial blood pressure of anesthetized rats. In guinea pig paired atria, Zo.Cr exhibited a cardiodepressant activity on the rate and force of spontaneous contractions. In rabbit thoracic aorta preparation, Zo.Cr relaxed the phenylephrine-induced vascular contraction at a dose 10 times higher than that required against K (80 mM)-induced contraction. Ca2+ channel-blocking (CCB) activity was confirmed when Zo.Cr shifted the Ca2+ dose-response curves to the right similar to the effect of verapamil. It also inhibited the phenylephrine (1 microM) control peaks in normal-Ca2+ and Ca2+-free solution, indicating that it acts at both the membrane-bound and the intracellular Ca2+ channels. When tested in endothelium-intact rat aorta, it again relaxed the K-induced contraction at a dose 14 times less than that required for relaxing the PE-induced contraction. The vasodilator effect of Zo.Cr was endothelium-independent because it was not blocked by L-NAME (0.1 mM) or atropine (1 microM) and also was reproduced in the endothelium-denuded preparations at the same dose range. These data indicate that the blood pressure-lowering effect of ginger is mediated through blockade of voltage-dependent calcium channels.

Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction

1. At the mouse neuromuscular junction, adenosine (AD) and the A(1) agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A(1) AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L- and N-type VDCCs), we measured the miniature end-plate potential (mepp) frequency in mouse diaphragm muscles. 2. Blockade of VDCCs by Cd(2+) prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not omega-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3. As A(1) receptors are coupled to a G(i/o) protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4. The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester epsilon6TDelta-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca(2+)-calmodulin. 5. To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K(+) concentrations. The effect of CCPA on ACh secretion evoked by 10 mm K(+) was prevented by the P/Q-type VDCC antagonist omega-agatoxin IVA. 6. CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20 mm K(+). We demonstrated that, at high K(+) concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A(1) receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mm K(+) in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K(+) concentrations, the activation of A(1) receptors by endogenous AD prevents excessive neurotransmitter release.

Action potential-independent release of glutamate by Ca2+ entry through presynaptic P2X receptors elicits postsynaptic firing in the brainstem autonomic network.

P2X receptors are ATP-gated channels permeable to cations including Ca(2+). In acute slices containing the nucleus of the solitary tract, in which neuronal ATP release and ATP-elicited physiological responses are demonstrated in vivo, we recorded spontaneous action potential-independent EPSCs [miniature EPSCs (mEPSCs)]. Activation of presynaptic P2X receptors with alpha,beta-methylene ATP (alphabetamATP) triggered Ca(2+)-dependent glutamate release that was resistant to blockade of voltage-dependent calcium channels but abolished by P2X receptor antagonists. mEPSCs elicited with alphabetamATP were of larger amplitude than basal mEPSCs and resulted in postsynaptic firing caused by temporal summation of miniature events. The large-amplitude mEPSCs provoked by alphabetamATP were likely to result from highly synchronized multivesicular release of glutamate at single release sites. Neither alphabetamATP nor ATP facilitated GABA release. We conclude that this facilitated release and consequent postsynaptic firing underlie the profound autonomic responses to activation of P2X receptors observed in vivo.

Unraveling Internalization Signals

Du et al . investigated the possibility that neuronal activity could influence internalization of neurotrophin-receptor complexes, a process required for some aspects of neurotrophin signaling, and discovered an unexpected link between calcium, receptor tyrosine kinase activity, and brain-derived neurotrophic factor (BDNF)-TrkB receptor complex internalization. Neuronal activity can promote the efficacy with which BDNF affects neuronal survival, synaptic efficacy, and synaptic plasticity, which provides a possible mechanism for selective activity-dependent enhancement of particular neuronal pathways. Du et al . used fluorescent imaging of biotinylated BDNF, quantification of radiolabeled BDNF on cell surface and the amount internalized, and Western analysis of TrkB in combination with electrical field stimulation to show that stimulus protocols associated with production of long-term potentiation promoted internalization of BDNF and TrkB in cultured hippocampal neurons. The authors used electrical stimulation of cells undergoing pharmacological blockade of voltage-dependent calcium channels or N -methyl-D-aspartate receptors, or in medium made without calcium, to show that activity-dependent internalization of BDNF and TrkB depended on calcium influx. BDNF stimulated tyrosine phosphorylation of TrkB, and electrical stimulation enhanced BDNF-dependent TrkB phosphorylation, an enhancement that depended on calcium influx. Pharmacological blockade of BDNF-dependent TrkB internalization did not affect activity-dependent enhancement of TrkB phosphorylation, whereas inhibition of TrkB kinase activity, either pharmacologically or with a dominant-negative TrkB mutant, blocked receptor internalization. Thus, neuronal activity appears to enhance BDNF-TrkB internalization through calcium-dependent stimulation of TrkB tyrosine phosphorylation. J. Du, L. Feng, E. Zaitsev, H.-S. Je, X.-W. Liu, B. Lu, Regulation of TrkB receptor tyrosine kinase and its internalization by neuronal activity and Ca 2+ influx. J. Cell Biol. 163 , 385-395 (2003). [Abstract] [Full Text]

Differential Effect of TEA on Long-Term Synaptic Modification in Hippocampal CA1 and Dentate Gyrus in vitro

The effectiveness of tetraethylammonium (TEA) and high-frequency stimulation (HFS) in inducing long-term synaptic modification is compared in CA1 and dentate gyrus (DG) in vitro. High-frequency stimulation induces long-term potentiation (LTP) at synapses of both perforant path-DG granule cell and Schaffer collateral-CA1 pyramidal cell pathways. By contrast, TEA (25 mM) induces long-term depression in DG while inducing LTP in CA1. The mechanisms underlying the differential effect of TEA in CA1 and DG were investigated. It was observed that T-type voltage-dependent calcium channel (VDCC) blocker, Ni2+ (50 μM), partially blocked TEA-induced LTP in CA1. A complete blockade of the TEA-induced LTP occurred when Ni2+ was applied together with the NMDA receptor antagonist, D-APV. The L-type VDCC blocker, nifidipine (20 μM), had no effect on CA1 TEA-induced LTP. In DG of the same slice, TEA actually induced long-term depression (LTD) instead of LTP, an effect that was blocked by D-APV. Neither T-type nor L-type VDCC blockade could prevent this LTD. When the calcium concentration in the perfusion medium was increased, TEA induced a weak LTP in DG that was blocked by Ni2+. During exposure to TEA, the magnitude of field EPSPs was increased in both CA1 and DG, but the increase was substantially greater in CA1. Tetraethylammonium application also was associated with a large, late EPSP component in CA1 that persisted even after severing the connections between CA3 and CA1. All of the TEA effects in CA1, however, were dramatically reduced by Ni2+. The results of this study indicate that TEA indirectly acts via both T-type VDCCs and NMDA receptors in CA1 and, as a consequence, induces LTP. By contrast, TEA indirectly acts via only NMDA receptors in DG and results in LTD. The results raise the possibility of a major synaptic difference in the density and/or distribution of T-type VDCCs and NMDA receptors in CA1 and DG of the rat hippocampus.

Calcium Influx through NMDA Receptors Directly Evokes GABA Release in Olfactory Bulb Granule Cells

Recurrent inhibition in olfactory bulb mitral cells is mediated via reciprocal dendrodendritic synapses with granule cells. Although GABAergic granule cells express both NMDA and non-NMDA glutamate receptors, dendrodendritic inhibition (DDI) relies on the activation of NMDA receptors. Using whole-cell recordings from rat olfactory bulb slices, we now show that olfactory NMDA receptors have a dual role; they depolarize granule cell spines, and they provide a source of calcium that can evoke GABA exocytosis. We demonstrate that exogenous NMDA can trigger GABA release after blockade of voltage-dependent calcium channels (VDCCs) with Cd. We also find that postsynaptic depolarization alone can evoke GABA release via a separate mechanism that relies on calcium influx through Cd-sensitive VDCCs. By selectively manipulating postsynaptic responses in granule cells with high-K or low-Na extracellular solutions, we show that endogenous glutamate can elicit GABA release via both NMDA receptor- and VDCC-dependent pathways. Finally, we find that blockade of Na channels in granule cells with tetrodotoxin enhances DDI, presumably by reducing the depolarization of granule cells during DDI and thereby increasing the driving force for Ca entry through NMDA receptors. These results provide evidence of a novel mechanism for evoked transmitter release that depends on Ca influx through ionotropic receptors and provides a new potential site for synaptic plasticity in the olfactory bulb.

Mechanisms of Cannabinoid Inhibition of GABAASynaptic Transmission in the Hippocampus

The localization of cannabinoid (CB) receptors to GABAergic interneurons in the hippocampus indicates that CBs may modulate GABAergic function and thereby mediate some of the disruptive effects of marijuana on spatial memory and sensory processing. To investigate the possible mechanisms through which CB receptors may modulate GABAergic neurotransmission in the hippocampus, whole-cell voltage-clamp recordings were performed on CA1 pyramidal neurons in rat brain slices. Stimulus-evoked GABA(A) receptor-mediated IPSCs were reduced in a concentration-dependent manner by the CB receptor agonist WIN 55,212-2 (EC(50) of 138 nM). This effect was blocked by the CB1 receptor antagonist SR141716A (1 microM) but not by the opioid antagonist naloxone. In contrast, evoked GABA(B)-mediated IPSCs were insensitive to the CB agonist. WIN 55,212-2 also reduced the frequency of spontaneous, action potential-dependent IPSCs (sIPSCs), without altering action potential-independent miniature IPSCs (mIPSCs), measured while sodium channels were blocked by tetrodotoxin (TTX). Blockade of voltage-dependent calcium channels (VDCCs) by cadmium also eliminated the effect of WIN 55,212-2 on sIPSCs. Depolarization of inhibitory terminals with elevated extracellular potassium caused a large increase in the frequency of mIPSCs that was inhibited by both cadmium and WIN 55,212-2. The presynaptic effect of WIN 55,212-2 was also investigated using the potassium channel blockers barium and 4-aminopyridine. Neither of these agents significantly altered the effect of WIN 55,212-2 on evoked IPSCs. Together, these data suggest that presynaptic CB1 receptors reduce GABA(A)- but not GABA(B)-mediated synaptic inhibition of CA1 pyramidal neurons by inhibiting VDCCs located on inhibitory nerve terminals.

Chronic nitric oxide inhibition model six years on.

The discovery in 1987 that endothelium-derived nitric oxide (NO) mediates the vasodilatory effect of certain endothelium-dependent agonists1 2 inaugurated the current huge field of NO biology. It is now recognized that NO plays essential roles in many diverse physiological processes and in some pathophysiologic events. Development of these concepts has been based largely on evidence obtained by limiting NO biosynthesis. This review is centered on the cardiovascular and particularly the renal functional and structural consequences of chronic pharmacologic NO inhibition by l-arginine analogues. We devoted special attention to the mechanisms of hypertension and organ injury that occur under these circumstances, while appreciating the inherent limitations surrounding interpretation of this data. NO is made by the enzymatic action of several widely distributed NO synthases (NOS). In the presence of the substrates l-arginine and oxygen, as well as a number of essential cofactors, NO is produced in response to appropriate stimuli. The constitutively expressed NOS play a major role in the physiological control of vascular tone and kidney function.3 4 Vascular endothelial NOS (eNOS) and brain-type NOS (bNOS) are widely distributed throughout the kidney5 as well as the cardiovascular system and in strategic locations in the peripheral and central nervous system (CNS).6 7 Both eNOS and particularly bNOS are abundant in the kidney, glomeruli, and vasculature as well as in most segments of the tubule,3 5 and NOS activity in medulla is considerably greater than in cortex.8 NO generated within the kidney controls the glomerular filtration rate (GFR), total renal and medullary blood flow, pressure natriuresis, epithelial sodium transport, and production of various vasoactive factors including renin.3 4 5 eNOS is distributed throughout most parts of the arterial and venous circulation, although there is considerable heterogeneity in the extent to which NO controls …

Phorbol ester and calcium regulation of corticotrophin-releasing factor receptor 1 expression in a neuronal cell line.

We have previously demonstrated that corticotrophin-releasing factor receptor 1 (CRF-R1) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, resulted in a time- and concentration-dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4alpha-phorbol failed to influence significantly CRF-R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down-regulated CRF-R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calcium channels with nifedipine inhibited agonist-mediated down-regulation of CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regulation of CRF-R1 mRNA levels and that calcium is required for agonist-mediated down-regulation of this receptor.

Action of angiotensin II, 5‐hydroxytryptamine and adenosine triphosphate on ionic currents in single ear artery cells of the rabbit

1. Angiotensin II, 5-hydroxytryptamine (5-HT) and adenosine triphosphate (ATP) evoked a transient inward current in isolated single car artery cells of rabbit held at -60 mV by whole cell voltage clamp in physiological saline using a KCL-containing pipette solution. Under these conditions agonist did not activate a calcium-dependent potassium current. 2. Responses to each agonist were transient and desensitized rapidly. Inward current at -60 mV holding potential was not abolished by blockade of voltage-dependent calcium channels or by buffering intracellular calcium with BAPTA, a calcium chelator, or following depletion of intracellular calcium stores with ryanodine. 3. The shape of the current-voltage relationships and the reversal potentials of the current induced by angiotensin II, 5-HT and ATP were similar under a variety of ionic conditions. Agonist-induced current was unaffected by replacing intracellular chloride with citrate ions or by replacing intracellular sodium with caesium or extracellular sodium with barium or calcium. Replacement of extracellular sodium with Tris shifted the reversal potential in all cases by around 30 mV negatively. 4. These data suggest that angiotensin II, 5-HT and ATP activate similar cationic conductances which are relatively non-selective allowing mono- and divalent cations to cross the smooth muscle cell membrane. These channels may allow the influx of calcium under physiological conditions.