Inhibition Of N-type Calcium Channels Research Articles

A Single Gβ Subunit Locus Controls Cross-talk between Protein Kinase C and G Protein Regulation of N-type Calcium Channels

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gbetagamma subunits in a G protein beta-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gbetagamma target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gbeta(1)-mediated responses. The molecular mechanisms that control this G protein beta subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent approximately 20-amino acid regions of the Gbeta subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gbetagamma signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase beta(2), and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gbeta(1) subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel alpha(1) subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel alpha(1) subunit.

Agonist-independent modulation of N-type calcium channels by ORL1 receptors.

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel-ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.

Cysteine String Protein (CSP) Inhibition of N-type Calcium Channels Is Blocked by Mutant Huntingtin

Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. We have previously reported interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels. These associations give rise to a tonic G protein inhibition of the channels. Here we have examined the effects of huntingtin fragments (exon 1) with (huntingtin(exon1/exp)) and without (huntingtin(exon1/nonexp)) expanded polyglutamine (polyQ) tracts on the CSP chaperone system. In vitro huntingtin(exon1/exp) sequestered CSP and blocked the association of CSP with G proteins. In contrast, huntingtin(exon1/nonexp) did not interact with CSP and did not alter the CSP/G protein association. Similarly, co-expression of huntingtin(exon1/exp) with CSP and N-type calcium channels eliminated CSP's tonic G protein inhibition of the channels, while coexpression of huntingtin(exon1/nonexp) did not alter the robust inhibition promoted by CSP. These results indicate that CSP's modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts.

Scrapie-infected GT1-1 cells show impaired function of voltage-gated N-type calcium channels (Ca v 2.2) which is ameliorated by quinacrine treatment

Prions are transmissible pathogens that cause neurodegenerative diseases, although the mechanisms behind the nervous system dysfunctions are unclear. To study the effects of a prion infection on voltage-gated calcium channels, scrapie-infected gonadotropin-releasing hormone neuronal cells (ScGT1-1) in culture were depolarized by KCl and calcium responses recorded. Lower calcium responses were observed in infected compared to uninfected cells. This effect was still observed when L-type calcium channels were blocked by nimodipine. After inhibition of N-type calcium channels with ω-conotoxin GVIA, there was no difference in calcium responses. The calcium responses after nimodipine treatment became progressively lower during infection, but there was no major loss of the cellular prion protein (PrP C) or marked increase in accumulation of the abnormal prion protein (PrP Sc) in the cultures. These results indicate that scrapie infection causes a dysfunction of voltage-gated N-type calcium channels, which is exacerbated slowly over time. Quinacrine treatment cleared PrP Sc and restored calcium responses in the ScGT1-1 cultures.

Mechanism of action of Gq to inhibit G beta gamma modulation of CaV2.2 calcium channels: probed by the use of receptor-G alpha tandems.

The stable interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the alpha(2A) adrenergic receptor (alpha(2A)-R) and pertussis toxin-resistant mutants of different G alpha subunits of heterotrimeric G-proteins. Both alpha(2A)-R-G alpha(o) and alpha(2A)-R-G alpha(i) proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (Ca(V)2.2) similar to the wild-type alpha(2A)-R when expressed in COS-7 cells. The interaction of G(q) with the G(i/o) signaling pathways was studied by expressing either G alpha(q) or a chimeric construct based on G alpha(q) containing the last five amino acids of G alpha(z), which is activated by alpha(2A)-R. It was found that G alpha(qz5) activated by the wild-type alpha(2A)-R inhibited Ca(V)2.2 currents in a voltage-independent fashion. Furthermore, G alpha(qz5) counteracted the voltage-dependent inhibition resulting from alpha(2A)-R-G alpha(o) activation. We subsequently investigated the basis for the behavior of G alpha(qz5). Our evidence suggests that this occurs as a result of a downstream effect of activation of G alpha(qz5) because it was blocked by C-terminal construct of phospholipase C beta 1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of G alpha(qz5) in alpha(2A)-R-G alpha(o)-transfected cells. Conversely, cells expressing both alpha(2A)-R-G alpha(o) and G alpha(qz5) exhibited a partial restoration of voltage-dependent inhibition in the presence of the PKC inhibitor bisindolylmaleimide I (GF 109203X). The potential sites of phosphorylation are discussed.

Slow inhibition of N-type calcium channels with GTP gamma S reflects the basal G protein-GDP turnover rate.

The inhibition of N-type Ca channels via a G protein pathway is a rapid mechanism for modulating Ca influx. It has been noted, however, that when G proteins are activated by guanosine 5'- O-(3-thiotriphosphate) (GTPgammaS), the speed of inhibition is greatly reduced, despite the pathway having fewer molecular steps. We explored this anomaly in chick dorsal root ganglion neurons by comparing Ca current inhibition using GTPgammaS with application of the G protein receptor agonist noradrenaline. Noradrenaline caused rapid Ca channel inhibition (tau~5 s), contrasting greatly with the ~70-fold slower rate observed with GTPgammaS. Additionally, the slow rate with GTPgammaS could be accelerated to near agonist-induced rates by application of noradrenaline, demonstrating that the inhibition with GTPgammaS was not perfusion limited and that the rate-limiting step was upstream from GTPgammaS binding. Our results suggest that in the absence of noradrenaline, G protein activation by GTPgammaS is impeded by the slow resting turnover of GDP/GTP. The rate at which inhibition develops with GTPgammaS (tau~350 s) is thus a direct and sensitive measure of resting GDP turnover.

Activation of group II mGlu receptors inhibits voltage-gated Ca2+ currents in myenteric neurons.

The enteric nervous system (ENS) contains functional ionotropic and group I metabotropic glutamate (mGlu) receptors. In this study, we determined whether enteric neurons express group II mGlu receptors and the effects of mGlu receptor activation on voltage-gated Ca(2+) currents in these cells. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a group II mGlu receptor agonist, reversibly suppressed the Ba(2+) current in myenteric neurons isolated from the guinea pig ileum. Significant inhibition was also produced by L-glutamate and the group II mGlu receptor agonists, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I), with a rank order potency of 2R,4R-APDC > DCG-IV > L-glutamate > L-CCG-I, and was reduced by the group II mGlu receptor antagonist LY-341495. Pretreatment of neurons with pertussis toxin (PTX) reduced the action of mGlu receptor agonists, suggesting participation of G(i)/G(o) proteins. Finally, omega-conotoxin GVIA blocked current suppression by DCG-IV, suggesting modulation of N-type calcium channels. mGlu2/3 receptor immunoreactivity was displayed by neurons in culture and in the submucosal and myenteric plexus of the ileum. A subset of these cells displayed a glutamatergic phenotype as shown by the expression of vesicular glutamate transporter 2. These results provide the first evidence for functional group II mGlu receptors in the ENS and show that these receptors are PTX sensitive and negatively coupled to N-type calcium channels. Inhibition of N-type calcium channels produced by activation of group II mGlu receptors may modulate enteric neurotransmission.

The novel N-type calcium channel blocker, AM336, produces potent dose-dependent antinociception after intrathecal dosing in rats and inhibits substance P release in rat spinal cord slices

N-type calcium channels modulate the release of key pro-nociceptive neurotransmitters such as glutamate and substance P (SP) in the central nervous system. Considerable research interest has focused on the therapeutic potential of the peptidic ω-conopeptides, GVIA and MVIIA as novel analgesic agents, due to their potent inhibition of N-type calcium channels. Recently, the novel peptidic N-type calcium channel blocker, AM336, was isolated from the venom of the cone snail, Conus catus. Thus, the aims of this study were to (i) document the antinociceptive effects of AM336 (also known as CVID) relative to MVIIA following intrathecal (i.t.) bolus dosing in rats with adjuvant-induced chronic inflammatory pain of the right hindpaw and to (ii) quantify the inhibitory effects of AM336 relative to MVIIA on K +-evoked SP release from slices of rat spinal cord. Both AM336 and MVIIA inhibited the K +-evoked release of the pro-nociceptive neurotransmitter, SP, from rat spinal cord slices in a concentration-dependent manner (EC 50 values=21.1 and 62.9 nM, respectively), consistent with the antinociceptive actions of ω-conopeptides. Following acute i.t. dosing, AM336 evoked dose-dependent antinociception (ED 50≈0.110 nmol) but the doses required to produce side-effects were an order of magnitude larger than the doses required to produce antinociception. For i.t. doses of MVIIA≤0.07 nmol, dose-dependent antinociception was also produced (ED 50≈0.016 nmol). Unexpectedly, however, i.t. doses of MVIIA>0.07 nmol, produced a dose-dependent decrease in antinociception but the incidence and severity of the side-effects continued to increase for all doses of MVIIA investigated, suggesting that dose-titration with MVIIA in the clinical setting, may be difficult.

Calcium Channel β Subunits Differentially Regulate the Inhibition of N-type Channels by Individual Gβ Isoforms

The direct inhibition of N- and P/Q-type calcium channels by G protein betagamma subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein beta subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. (2000) J. Physiol. (Lond.) 527, 203-212; Cooper, C. B., Arnot, M. I., Feng, Z.-P., Jarvis, S. E., Hamid, J., Zamponi, G. W. (2000) J. Biol. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel beta subunits to modulate the inhibition of alpha(1B) N-type calcium channels by the five known different Gbeta subunit subtypes. Our data reveal that the degree of inhibition by a particular Gbeta subunit is strongly dependent on the specific calcium channel beta subunit, with N-type channels containing the beta(4) subunit being less susceptible to Gbetagamma-induced inhibition. The calcium channel beta(2a) subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For Gbeta(3)-mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the beta(2a) N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel beta subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel beta subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.

Nitric oxide-cGMP pathway facilitates acetylcholine release and bradycardia during vagal nerve stimulation in the guinea-pig in vitro.

1. We tested the hypothesis that nitric oxide (NO) augments vagal neurotransmission and bradycardia via phosphorylation of presynaptic calcium channels to increase vesicular release of acetylcholine. 2. The effects of enzyme inhibitors and calcium channel blockers on the actions of the NO donor sodium nitroprusside (SNP) were evaluated in isolated guinea-pig atrial-right vagal nerve preparations. 3. SNP (10 microM) augmented the heart rate response to vagal nerve stimulation but not to the acetylcholine analogue carbamylcholine (100 nM). SNP also increased the release of [3H]acetylcholine in response to field stimulation. No effect of SNP was observed on either the release of [3H] acetylcholine or the HR response to vagal nerve stimulation in the presence of the guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10 microM). 4. The phosphodiesterase 3 (PDE 3) inhibitor milrinone (1 microM) increased the release of [3H] acetylcholine and the vagal bradycardia and prevented any further increase by SNP. SNP was still able to augment the vagal bradycardia in the presence of the protein kinase G inhibitor KT5823 (1 microM) but not after protein kinase A (PKA) inhibition with H-89 (0.5 microM) or KT5720 (1 microM) had reduced the HR response to vagal nerve stimulation. Neither milrinone nor H-89 changed the HR response to carbamylcholine. 5. SNP had no effect on the magnitude of the vagal bradycardia after inhibition of N-type calcium channels with omega-conotoxin GVIA (100 nM). 6. These results suggests that NO acts presynaptically to facilitate vagal neurotransmission via a cGMP-PDE 3-dependent pathway leading to an increase in cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channels. This pathway may augment the HR response to vagal nerve stimulation by increasing presynaptic calcium influx and vesicular release of acetylcholine.

Distinct Molecular Determinants Govern Syntaxin 1A-Mediated Inactivation and G-Protein Inhibition of N-Type Calcium Channels

We have reported recently that syntaxin 1A mediates two effects on N-type channels transiently expressed in tsA-201 cells: a hyperpolarizing shift in the steady-state inactivation curve as well as a tonic inhibition of the channel by G-protein betagamma subunits (Jarvis et al., 2000). Here we have examined some of the molecular determinants and factors that modulate the action of syntaxin 1A on N-type calcium channels. With the additional coexpression of SNAP25, the syntaxin 1A-induced G-protein modulation of the channel became reduced in magnitude by approximately 50% but nonetheless remained significantly higher than the low levels of background inhibition seen with N-type channels alone. In contrast, coexpression of nSec-1 did not reduce the syntaxin 1A-mediated G-protein inhibition; however, interestingly, nSec-1 was able to induce tonic G-protein inhibition even in the absence of syntaxin 1A. Both SNAP25 and nSec-1 blocked the negative shift in half-inactivation potential that was induced by syntaxin 1A. Activation of protein kinase C via phorbol esters or site-directed mutagenesis of three putative PKC consensus sites in the syntaxin 1A binding region of the channel (S802, S896, S898) to glutamic acid (to mimic a permanently phosphorylated state) did not affect the syntaxin 1A-mediated G-protein modulation of the channel. However, in the S896E and S898E mutants, or after PKC-dependent phosphorylation of the wild-type channels, the susceptibility of the channel to undergo shifts in half-inactivation potential was removed. Thus, separate molecular determinants govern the ability of syntaxin 1A to affect N-type channel gating and its modulation by G-proteins.

Ca(2+) channels involved in the generation of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurons.

The advantages of using isolated cells have led us to develop short-term cultures of hippocampal pyramidal cells, which retain many of the properties of cells in acute preparations and in particular the ability to generate afterhyperpolarizations after a train of action potentials. Using perforated-patch recordings, both medium and slow afterhyperpolarization currents (mI(AHP) and sI(AHP), respectively) could be obtained from pyramidal cells that were cultured for 8-15 days. The sI(AHP) demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices. In addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have shown that the sI(AHP) is also insensitive to 100 nM charybdotoxin but is inhibited by 100 microM D-tubocurarine. Concentrations of nifedipine (10 microM) and nimodipine (3 microM) that maximally inhibit L-type calcium channels reduced the sI(AHP) by 30 and 50%, respectively. However, higher concentrations of nimodipine (10 microM) abolished the sI(AHP), which can be partially explained by an effect on action potentials. Both nifedipine and nimodipine at maximal concentrations were found to reduce the HVA calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, omega-conotoxin GVIA (100 nM), irreversibly inhibited the sI(AHP) by 37%. Together, omega-conotoxin (100 nM) and nifedipine (10 microM) inhibited the sI(AHP) by 70%. 10 microM ryanodine also reduced the sI(AHP) by 30%, suggesting a role for calcium-induced calcium release. It is concluded that activation of the sI(AHP) in cultured hippocampal pyramidal cells is mediated by a rise in intracellular calcium involving multiple pathways and not just entry via L-type calcium channels.

Adenosine A1 and class II metabotropic glutamate receptors mediate shared presynaptic inhibition of retinotectal transmission.

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that adenosine A1 receptors mediate presynaptic inhibition at the retinotectal synapse of goldfish. Here we extend these findings to metabotropic glutamate receptors (mGluRs) and report that presynaptic inhibition produced by both A1 adenosine receptors and group II mGluRs is due to G(i) protein coupling to inhibition of N-type calcium channels in the retinal ganglion cells. Adenosine (100 microM) and an A1 (but not A2) receptor agonist reduced calcium current (I(Ca2+)) by 16-19% in cultured retinal ganglion cells, consistent with their inhibition of retinotectal synaptic transmission (-30% amplitude of field potentials). The general metabotropic glutamate receptor (mGluR) agonist 1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 50 microM) and the selective group II mGluR receptor agonist (2S, 2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 300 nM) inhibited both synaptic transmission and I(Ca2+), whereas the group III mGluR agonist L-2-amino-4-phosphono-butyrate (L-AP4) inhibited neither synaptic transmission nor I(Ca2+). When the N-type calcium channels were blocked with omega-conotoxin GVIA, both adenosine and DCG-IV had much smaller percentage effects on the residual 20% of I(Ca2+), suggesting effects mainly on the N-type calcium channels. The inhibitory effects of A1 adenosine receptors and mGluRs were both blocked by pertussis toxin, indicating that they are mediated by either G(i) or G(o). They were also inhibited by activation of protein kinase C (PKC), which is known to phosphorylate and inhibit G(i). Finally, when applied sequentially, inhibition by adenosine and DCG-IV were not additive but occluded each other. Together these results suggest that adenosine A1 receptors and group II mGluRs mediate presynaptic inhibition of retinotectal synaptic transmission by sharing a pertussis toxin (PTX)-sensitive, PKC-regulated G(i) protein coupled to N-type calcium channels.

Endogenous Angiotensin II and Bradykinin Delay and Attenuate the Hypotension After N-Type Calcium Channel Blockade in Conscious Rabbits

The effects of N-type calcium channel inhibition with omega-conotoxin GVIA (omega-CTX) on cardiovascular parameters and vagally mediated autonomic reflexes and the role of the renin-angiotensin system were assessed in conscious rabbits. Omega-CTX (10 microg/kg, i.v.) resulted in hypotension, tachycardia, and attenuation of the sympathetic and vagal components of the baroreceptor-heart rate reflex (baroreflex). In the control group (no pretreatment), the peak decrease in mean arterial pressure (MAP) of 13 +/- 3 mm Hg from 72 +/- 2 mm Hg occurred after 33 +/- 3 min, with a corresponding tachycardia of 80 +/- 20 beats/min (n = 6). The tachycardia was due to vagal withdrawal, as a similar increase in heart rate (84 +/- 8 beats/min) after omega-CTX was observed after pretreatment with the beta-adrenoceptor antagonist, propranolol (n = 6). Angiotensin-converting enzyme (ACE) inhibition with enalaprilat revealed a larger, more rapid decrease in MAP in response to omega-CTX (-19 +/- 4 mm Hg from 65 +/- 1 mm Hg after 18 +/- 2 min; n = 6) compared with the control group. Similar larger decreases in MAP were also observed in the presence of the AT1-receptor antagonist, losartan, or the bradykinin B2 receptor antagonist, HOE-140 (n = 5-6). Pretreatment with enalaprilat, losartan, or HOE-140 caused a 50% decrease in the reflex tachycardia after omega-CTX compared with that observed in the control group, and omega-CTX caused a greater attenuation of the vagal component of the baroreflex and a decrease in the bradycardia evoked by the Bezold-Jarisch-like reflex. Also, there was a significant decrease in the bradycardia induced by the nasopharyngeal reflex after omega-CTX in the presence of ACE inhibition and HOE-140. Thus in the conscious rabbit, angiotensin II and bradykinin have a role in attenuating and slowing the hypotensive effect of N-type calcium channel inhibition. Vagolytic effects of omega-CTX on the baroreflex are augmented, and on other vagal reflexes are unmasked, via inhibition of the renin-angiotensin system. The complexity and mechanism of the interaction between N-type calcium channels and the renin-angiotensin system remain to be elucidated.

G-Protein β-Subunit Specificity in the Fast Membrane-Delimited Inhibition of Ca2+Channels

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.

Direct and indirect actions of 5-hydroxytryptamine on the discharge of mesenteric afferent fibres innervating the rat jejunum.

1. This study was performed to elucidate the actions of 5-hydroxytryptamine (5-HT) on mesenteric afferent discharge and to determine the receptor-mechanisms responsible for these effects. The activity of mesenteric afferents innervating the mid-jejunum of urethane-anaesthetized rats was recorded with extracellular microelectrodes. The discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software. 2. The intravenous injection of 5-HT produced a complex pattern of afferent activation with two distinct components which could be distinguished both in terms of the response characteristics and the receptors involved. Initially, in 64% of nerve bundles, there was a brief (2.0 +/- 0.1 s) but intense activation of afferent discharge with peak afferent firing increasing with incremental doses of 5-HT. The discharge frequency in seventeen single units from these bundles during the initial response to 10 micrograms 5-HT was 13.0 +/- 1.8 impulses s-1 from a baseline discharge of 1.0 +/- 0.1 impulses s-1. 3. This initial response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, whereas 5-methoxytryptamine (5-MEOT, 10-100 micrograms) had no comparable effect. Similarly, the initial 4. 5-HT response was completely abolished by the 5-HT3 receptor antagonist, granisetron (0.5 mg kg-1). 5-HT also evoked, in approximately 35% of nerve bundles, a delayed response that single unit analysis showed to be mediated by an entirely different population of afferents from those activated during the initial response. This secondary response to 5-HT was characterized by a more prolonged (> 30 s) but less intense period of afferent activity which was coincident with an increase in intrajejunal pressure, and was mimicked by 5-MEOT (10-100 micrograms). The secondary response to 5-HT and the response to 5-MEOT were significantly attenuated by the 5-HT2A receptor antagonist, ketanserin (0.5 mg kg-1), which had no effect on the initial response. The initial response to 5-HT was unaffected by the L-type calcium channel inhibitor nifedipine (1 mg kg-1) or the N-type calcium channel inhibitor omega-conotoxin GVIA (25 micrograms kg-1). However, the secondary response to 5-HT was significantly reduced after treatment with nifedipine. 5. These results demonstrate that 5-HT activates different populations of afferent fibres innervating the rat jejunum. One population of afferents is activated directly via stimulation of 5-HT3 receptors, while another population responds to 5-HT with a time course consistent with secondary activation of mechanosensitive afferents following 5-HT2A-mediated contractile activity.

Activation of adenosine A1 and A2 receptors differentially affects acetylcholine release from electric organ synaptosomes by modulating calcium channels

Adenosine inhibited the release of acetylcholine (ACh) evoked by high K + depolarization from synaptosomes isolated from the electric organ of the Japanese electric ray Narke japonica. The adenosine A 1 receptor agonist N 6-cyclohexyladenosine was an effective inhibitor. Conversely, in the presence of an A 1 receptor antagonist, 8-cyclopentyltheophylline, adenosine potentiated the release of ACh. The A 2 receptor agonist N 6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl] adenosine also facilitated the evoked ACh release. Thus, adenosine inhibits the evoked release of ACh via the A 1 receptor while it facilitates the release via the A 2 receptor. The EC 50 for inhibition and facilitation by adenosine was about 1 and 41 μM, respectively. There are three known types of calcium channels (N-, P/Q- and L-type) in synaptosomes. The effects of Ca 2+ channel type-specific blockers on the modulation of ACh release by adenosine A 1 or A 2 receptor activation revealed that inhibition by A 1 receptor activation was caused via inhibition of N-type calcium channels and the facilitative effects by A 2 receptor activation was mediated by potentiation of P-type calcium channels.

Calcineurin modulates G protein-mediated inhibition of N-type calcium channels in rat sympathetic neurons.

The modulation of N-type voltage-gated calcium (Ca2+) channels by G protein-coupled receptors was investigated in sympathetic neurons of the male rat major pelvic ganglion (MPG) with the use of whole cell patch-clamp recording techniques from acutely dissociated neurons. By inhibiting calcineurin, a Ca2+/calmodulin-regulated protein phosphatase, the alpha2 noradrenergic and somatostatin receptor-induced inhibition of these N-type Ca2+ channels was greatly reduced. Both of these receptor pathways utilize a pertussis toxin-sensitive G protein (G(PTX)). The guanosine 5'-o-(3-thiotriphosphate) (GTPgammaS)-induced decrease in the amplitude and activation kinetics of Ca2+ currents, an effect that was similar to the activation of G(PTX)-coupled receptors, also was reduced by the inhibition of calcineurin. Calcineurin does not regulate the muscarinic receptor-induced inhibition of the N-type Ca2+ channels, a pathway that utilizes a different G protein in the MPG neurons. Thus calcineurin appears to selectively regulate the coupling between the G(PTX) and the Ca2+ channel.

Bradykinin Inhibition of N- and L-type Calcium Channel Currents in NG108-15 Cells

The bradykinin regulation of calcium channel currents in NG108-15 neuroblastoma × glioma hybrid cells was examined, in order to determine: (1) which type of bradykinin receptor mediates the inhibition of N-type calcium channels in these cells; and (2) whether bradykinin can modulate other types of calcium channels in these cells. Bradykinin inhibited both N- and L-type calcium channels in NG108-15 cells, with ec50s of 10 ± 2 nM and 29 ± 7 nM, respectively. The inhibition of both L- and N-type calcium channels by bradykinin (100 nM) could be completely inhibited by the bradykinin B2 receptor antagonist Hoe 140 (10 nM). Bradykinin appeared to inhibit that portion of the L-type calcium channel current that was also reversibly inhibited by ω-conotoxin GVIA. The bradykinin inhibition of the L-type calcium channel current was partly reduced by pretreatment of the cells with pertussis toxin, whereas the inhibition of the N-type current was pertussis toxin-insensitive. In some cultures it was observed that the bradykinin B1 receptor agonist desArg9bradykinin inhibited the L-type calcium channel current. © 1997 Elsevier Science Ltd. All rights reserved.

Elementary events underlying voltage-dependent G-protein inhibition of N-type calcium channels

Voltage-dependent G-protein inhibition of N-type calcium channels reduces presynaptic calcium entry, sharply attenuating neurotransmitter release. Studies in neurons demonstrate that G-proteins have multiple modulatory effects on N-type channels. The observed changes may reflect genuine complexity in G-protein action and/or the intricate interactions of multiple channels and receptors in neurons. Expression of recombinant M2-muscarinic receptors and N-type channels in HEK 293 cells allowed voltage-dependent inhibition to be studied in isolation. In this system, receptor-activated G-proteins had only one effect: a 10-fold increase in the time required for channels to first open following membrane depolarization. There were no changes in gating after the channel first opened, and unitary currents were not detectably altered by modulation. Despite its simplicity, this single change successfully accounts for the complex alterations in whole-cell current observed during G-protein inhibition in neurons.