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CARDIOVASCULAR
-Dependent Inhibition of Voltage-Operated Ca2+ ChannelsDepartments of Pharmacology (C.M., R.E., G.W.A., R.L.) and Medicine, Greenberg Division of Cardiology (G.W.A.), Weill Cornell Medical College, New York, New York
Received for publication
February 11, 2008
Accepted
June 2, 2008.
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Abstract |
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i impairs the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, and this decreases Ca2+ influx via voltage-operated Ca2+ channels (VOCC). Yet, the Gi/o-derived β
dimer could directly inhibit VOCC, and the subsequent reduction in Ca2+ influx would be responsible for the H3R-mediated attenuation of transmitter exocytosis. In this study, we tested this hypothesis in nerve-growth factor-differentiated rat pheochromocytoma cells (PC12) stably transfected with H3R (PC12-H3) and with the Gβ scavenger β-adrenergic receptor kinase 1 (β-ARK1)-(495-689)-polypeptide (PC12-H3/β-ARK1). Thus, we evaluated the effects of H3R activation directly on the following: 1) Ca2+ current (ICa) using the whole-cell patch-clamp technique; and 2) K+-induced exocytosis of endogenous dopamine. H3R activation attenuated both peak ICa and dopamine exocytosis in PC12-H3 but not in PC12-H3/β-ARK1 cells. Moreover, a membrane permeable phosducin-like Gβ scavenger also prevented the antiexocytotic effect of H3R activation. In contrast, the H3R-induced attenuation of cAMP accumulation and dopamine exocytosis in response to forskolin were the same in both PC12-H3 and PC12-H3/β-ARK1 cells. Our findings reveal that although Gi participates in the H3-mediated antiexocytotic effect when the adenylyl cyclase-cAMP-PKA pathway is stimulated, a direct Gβ-induced inhibition of VOCC, resulting in an attenuation of ICa, plays a pivotal role in the H3R-mediated decrease in [Ca2+]i and associated cardioprotective antiexocytotic effects. The discovery of this H3R-signaling step may offer new therapeutic approaches to cardiovascular diseases characterized by hyperadrenergic activity.
), guinea pig (Endou et al., 1994), and human hearts (Imamura et al., 1995) express histamine H3-receptors (H3R). H3R activation results in attenuation of excessive norepinephrine release in myocardial ischemia, a recognized cardioprotective effect, because it alleviates arrhythmic cardiac dysfunction (Levi and Smith, 2000).
We previously reported that imetit, a selective H3R agonist (Garbarg et al., 1992), reduces norepinephrine exocytosis evoked by depolarization of cardiac sympathetic nerve endings (Imamura et al., 1994; Imamura et al., 1995), an action associated with a marked decrease in intraneuronal Ca2+ ([Ca2+]i) (Silver et al., 2002). It is conceivable that H3R activation may decrease [Ca2+]i by inhibiting Ca2+ influx through voltage-operated Ca2+ channels (VOCC) in sympathetic nerve terminals. H3R-mediated inhibition of N-type Ca2+ channel current has been claimed to occur in histaminergic neurons from the rat hypothalamus; an unverified claim, however, because selective H3R antagonists were not shown to block this effect (Takeshita et al., 1998).
Although N-type Ca2+ channels are the dominant Ca2+ entry pathway triggering sympathetic transmitter release (Lipscombe et al., 1989; Zhu and Yakel, 1997), it is possible that entry of Ca2+ through L-type Ca2+ channels may also be important in norepinephrine exocytosis and be inhibited by H3R activation. Indeed, we found that H3R activation synergizes with both N- and L-type Ca2+ channel blockers to reduce K+-induced norepinephrine release from cardiac synaptosomes (Seyedi et al., 2005).
H3R-mediated attenuation of norepinephrine exocytosis from cardiac sympathetic nerves involves an H3R-Gi/o coupling, adenylyl cyclase inhibition by Gi, decreased cAMP formation, and diminished PKA activity (Seyedi et al., 2005). Inasmuch as a decrease in PKA activity is likely to decrease phosphorylation of VOCC, which would lead to a decrease in voltage-activated calcium current (ICa), it is plausible that the H3R-mediated decrease in norepinephrine exocytosis results from a decreased Ca2+ influx via VOCC due to diminished activity of the adenylyl cyclase-cAMP-PKA pathway.
On the other hand, Gβ is known to decrease adenylyl cyclase activity (Taussig et al., 1993), and it is therefore possible that in addition to the attenuation of adenylyl cyclase by Gi (Seyedi et al., 2005), Gβ will also play a role in the H3R-mediated decrease in cAMP. Yet, because the Gβ dimer is known to directly inhibit VOCC (Herlitze et al., 1996; Ikeda, 1996), we questioned whether the H3R-mediated attenuation of norepinephrine exocytosis may also result from an inhibition of Ca2+ influx by a Gβ action.
Accordingly, the purpose of our investigation was to determine the role of a Gβ-induced inhibition of VOCC in the H3R-mediated attenuation of norepinephrine exocytosis. To accomplish this, we evaluated the effects of H3R activation on ICa and endogenous transmitter exocytosis in nerve-growth factor (NGF)-differentiated rat pheochromocytoma cells (PC12) stably transfected with H3R and the Gβ scavenger β-adrenergic receptor kinase 1 (β-ARK1)-(495-689)-polypeptide (Koch et al., 1994; Dickenson and Hill, 1998). NGF-differentiated PC12 cells were chosen as a functional model of sympathetic neurotransmission because their phenotype closely resembles that of sympathetic neurons (Dichter et al., 1977; Taupenot, 2007). Our results demonstrate a pivotal involvement of the Gβ subunit in H3R-mediated attenuation of neuronal ICa and consequent neurotransmitter exocytosis.
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Materials and Methods |
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Reverse-Transcriptase Polymerase Chain Reaction. Total RNA was isolated from PC12 and PC12-H3 cells using the TRIzol RNA purification kit (Invitrogen). cDNAs were synthesized from total RNA using Superscript II reverse transcriptase (Invitrogen) and random primers as described by the manufacturer. Polymerase chain reaction (PCR) was used to detect the H3 mRNA expression with cDNA from either PC12 or PC12-H3 as templates, and with P1: 5'-CTCTGCAAGCTGTGGCTGGTGGTAGACTACCTACTGTGTG-3' and P2: 5'-CTTCTTGTCCCGCGACAGCCGAAAGCGCTGGGTGATGCTT-3' as primers (Invitrogen). PCRs were performed under the following conditions: 94°C for 40 s; 65°C for 40 s; and 72°C for 2 min, for 40 cycles. As a control, total HeLa RNA was used as the template for amplification of a 353-base pairs (bp) segment of β-actin mRNA in a parallel PCR reaction. The PCR products were run on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light.
Western Blotting. Cell lysates (20 µg) prepared from PC12-H3 and PC12-H3/β-ARK1 cells were separated by SDS-polyacrylamide gel electrophoresis 10 to 20% acrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA). After transfer, the membrane was washed with Tris-buffered saline (TBS) and blocked for 2 h at room temperature in blocking buffer (TBS containing 0.1% Tween 20 and 5% (w/v) nonfat dry milk). Blots were then incubated overnight at 4°C with anti-β-ARK1 primary antibody (Epitomics, Burlingame, CA) at 1:500 dilution in 5% (w/v) bovine serum albumin dissolved in TBS-Tween 20 (0.1%). The primary antibody was removed, and the blot was extensively washed with TBS/Tween 20. Blots were then incubated for 2 h at room temperature with horseradish peroxidase-coupled anti-rabbit IgG (Cell Signaling Technology, Inc., Danvers, MA) at a 1:3000 dilution in blocking buffer. After removal of the secondary antibody, blots were extensively washed as described above and developed using the enhanced chemiluminescence detection system (Pierce, Rockford, IL) followed by exposure to X-ray film (Biomax MR; Care-stream Health, Rochester, NY).
cAMP Measurement. Cellular cAMP accumulation was measured in PC12-H3 and PC12-H3/β-ARK1 cells seeded in 96-well plates and differentiated with NGF (100 ng/ml) for 5 to 7 days. After a 20-min treatment with the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (2 mM), cells were incubated for 5 min with or without the H3R agonist imetit (100 nM), either alone or in combination with the H3R antagonist clobenpropit (CBP) (50 nM). Cells were incubated with CBP for 5 min before the addition of imetit. Intracellular cAMP levels were then enhanced with forskolin (10 µM) for 20 min. The incubation buffer was immediately aspirated, cells were lysed, and intracellular cAMP levels were determined using a cAMP Biotrak EIA kit (GE Healthcare, Chalfont St. Giles, UK) following the manufacturer's protocol. All drugs were constituted in HEPES-buffered Na+ Ringer's solution (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl2, and 1 mM MgCl2, pH 7.4).
Dopamine Assay. PC12-H3 and PC12-H3/β-ARK1 cells cultured in 12-well plates and differentiated with NGF (100 ng/ml) for 5 to 7 days were incubated with drugs for 5 min. When CBP or the anti-β peptide was used, cells were incubated with these compounds for 5 min before incubation with imetit. For pertussis toxin (PTX) treatment, cells were incubated for 24 h with PTX (200 ng/ml) before assay. Dopamine exocytosis was elicited by incubating samples for 5 min with K+ 100 mM (osmolarity was maintained constant by adjusting the NaCl concentration), phorbol 12-myristate 13-acetate (PMA; 300 nM), or for 20 min with forskolin (10 µM). At the end of the incubation period, aliquots of the supernatant and cell lysates (after a 30-min treatment with 0.3% Triton X-100) were taken from each well and analyzed for dopamine content by high-performance liquid chromatography with electrochemical detection as previously described (Seyedi et al., 2005) with a 6.0-min retention time.
Electrophysiology. Whole-cell voltage-clamp studies were performed on PC12 cells. The cells were transferred to 22 x 22-mm glass coverslips coated with poly-L-lysine and collagen and differentiated with NGF (50 ng/ml) for 3 to 7 days before use in voltage-clamp experiments. Bath solution was as follows: 20 mM BaCl2, 125 mM N-methyl-D-glucamine, and 10 mM HEPES, pH 7.5. Pipettes had resistances of 6 to 9 M
when filled with intracellular solution containing the following: 100 mM N-methyl-D-glucamine, 20 mM TEACl, 10 mM EGTA, 2 mM MgCl2, 10 mM glucose, 2 mM Na2ATP, and 10 mM HEPES, pH 7.35. Fragments of coverslip were transferred to a recording chamber, and the medium was rinsed off with bath solution. After adoption of whole-cell configuration, currents were recorded either in the absence (baseline) or presence of drugs. Cells were held at -40 mV and subjected to 200-ms test pulses from -40 mV to +40 mV in 10-mV increments every 30 s. Recordings were performed at 22 to 25°C using an IX50 inverted microscope (Olympus, Tokyo, Japan), a Multiclamp 700A Amplifier, a Digidata 1300 Analog/Digital converter, and pClamp9 software (Molecular Devices, Sunnyvale, CA). Recordings were performed at 10-kHz sampling frequency, with no filtering before analysis, and were subsequently low-pass Bessel filtered for presentation purposes. All recordings with leak currents >20 pA or an access resistance >25 M were discarded.
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act).
Reagents. CBP, imetit, IBMX, NGF, 
-conotoxin GVIA (-CTX), PMA, and PTX were prepared in aqueous solution, whereas dimethyl sulfoxide was the vehicle for IBMX, forskolin, and nifedipine. The anti-β peptide was obtained from AnaSpec, Inc. (San Jose, CA), imetit was obtained from Tocris Bioscience (Ellisville, MO), and PMA was from LC Laboratories (Woburn, MA). All other chemicals were reagent grade and purchased from Sigma-Aldrich.
Statistics. cAMP levels are expressed as mean absolute values ± S.E.M. Dopamine release values are expressed as mean percentage increases above basal level ± S.E.M. Current density is expressed as mean ± S.E.M., with n specifying the number of independent experiments. Statistical significance was assessed by Student's t test or one-way analysis of variance (ANOVA) followed by post hoc testing (Dunnett's test) as indicated in the appropriate figure legend. Significance was asserted if p < 0.05.
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700 bp was amplified, demonstrating the presence of H3R in PC12-H3 cells; this band was absent in nontransfected PC12 cells (Fig. 1A). The transfected H3R were Gi/o-coupled as demonstrated by the loss of their antiexocytotic activity after pretreatment with PTX (100 ng/ml; Figs. 2C and 4). Stable expression of β-ARK1-(495-689)-polypeptide in PC12 cells was determined by Western blotting. A band of 27 kDa confirmed the expression of β-ARK1 in PC12-H3/β-ARK1 cells, but not in PC12-H3 cells (Fig. 1B).
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i (Lovenberg et al., 1999), we determined that H3R activation did indeed reduce the formation of cAMP in PC12-H3 cells in response to forskolin. In the presence of the selective agonist imetit (100 nM) (Garbarg et al., 1992), a 20-fold forskolin-induced (10 µM) increase in intracellular cAMP concentration was attenuated by 70% (Fig. 2A). Clobenpropit (50 nM), a selective H3R antagonist (Van der Goot et al., 1992), reduced the effect of imetit by 60% (Fig. 2A). This confirmed that the Gi/o-signaling cascade associated with transfected H3R was functional.
Inasmuch as some forms of adenylyl cyclase are inhibited by the Gβ dimer (Tang and Gilman, 1991), we questioned whether the decrease in intracellular cAMP concentration in the presence of imetit was due to a Gβ-induced diminution of cAMP-PKA activity. To evaluate this possibility, we determined cAMP accumulation and endogenous dopamine exocytosis (Chen and Westfall, 1994) in response to forskolin, in control and H3R-activated conditions, in PC12-H3 cells stably transfected with the Gβ scavenger β-ARK1 polypeptide (Koch et al., 1994) and compared them to those measured in PC12-H3 cells. In PC12-H3/β-ARK1 cells, forskolin (10 µM) elicited a 15-fold increase in intracellular cAMP concentration, which was comparable to that observed in PC12-H3 cells (Fig. 2, compare A and B). In the presence of imetit (100 nM), the increase in cAMP formation was attenuated by 70%; clobenpropit (50 nM) reduced the effect of imetit by 60% (Fig. 2B). Thus, H3R activation produced effects of similar magnitude in PC12-H3 and PC12-H3/β-ARK1 cells (Fig. 2, compare A and B), indicating that the Gβ complex does not have inhibitory effects on the form of adenylyl cyclase present in PC12 cells. Furthermore, forskolin (10 µM) elicited an 2- and 3-fold increase in dopamine release in PC12-H3 and PC12-H3/β-ARK1 cells, respectively (Fig. 2, C and D). H3R activation with imetit also significantly attenuated dopamine exocytosis (by 60%) in both PC12-H3 and PC12-H3/β-ARK1 cells; this effect was inhibited by H3R blockade with clobenpropit (Fig. 2, C and D). Thus, in differentiated PC12 cells stimulated with forskolin, the H3R-mediated decrease in cAMP and associated antiexocytotic effect derive mostly from an inhibition of adenylyl cyclase by Gi and not by Gβ.
We next measured peak ICa in PC12-H3 cells using the conventional whole-cell patch-clamp technique (Fig. 3). The ICa was activated at voltages equal or positive to -20 mV and reached a peak at +10 mV. Peak ICa was markedly reduced by incubation with -CTX (100 nM) or nifedipine (5 µM), indicating that both N- and L-type Ca2+ channels contribute to ICa in these cells (Fig. 3, A and E). Indeed, combined treatment of the cells with nifedipine and -CTX blocked 90% of the ICa (Fig. 3A). When PC12-H3 cells were incubated with imetit (100 nM), peak ICa was significantly attenuated; this response was antagonized by clobenpropit (50 nM) (Fig. 3, B, C, and E). Stimulation of adenylyl cyclase with forskolin (10 µM) enhanced peak ICa by 25%; again, this current was markedly reduced by imetit, and clobenpropit antagonized the effect of imetit (Fig. 3, D and E).
We next depolarized PC12-H3 cells with K+ (100 mM) and determined the exocytosis of endogenous dopamine. Depolarization with K+ elicited an 2.5-fold increase in dopamine release, a response curtailed by 70% by the L-type Ca2+ channel antagonist nifedipine (5 µM) but not by the N-type channel antagonist -CTX (100 nM) (Fig. 4). H3R activation with imetit also significantly attenuated dopamine exocytosis (30%); this effect was abolished by H3R blockade with clobenpropit, a further confirmation of the functionality of transfected H3R (see Fig. 2A). Thus, H3R activation attenuated both peak ICa and dopamine exocytosis, suggesting that H3R-mediated antiexocytotic effects could result from a reduced Ca2+ influx via VOCC.
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subunits are known to directly inhibit VOCC function (Herlitze et al., 1996; Ikeda, 1996), we next assessed whether the Gβ dimer plays a role in the decrease in ICa and associated antiexocytotic effect observed with H3R activation. Peak ICa in PC12-H3/β-ARK1 cells did not differ from that recorded in PC12-H3 cells (Fig. 5A), suggesting that Gβ was not constitutively active in these cells. In contrast to what was observed in PC12-H3 cells, imetit failed to reduce peak ICa in PC12-H3/β-ARK1 cells, indicating that the Gβ dimer plays a role in the H3R-mediated reduction of ICa (compare Figs. 3B and 5A). Furthermore, the slowing of current activation, as demonstrated by an increase in act in the presence of imetit, supports a direct Gβ-induced inhibition of VOCC (Fig. 3C) (Stephens et al., 1998). In addition, whereas the antiexocytotic effect of nifedipine was preserved in PC12-H3/β-ARK1 cells, imetit failed to affect dopamine exocytosis (Fig. 5B), and imetit did not modify dopamine exocytosis in PC12-H3 cells incubated with a membrane-permeable phosducin-like anti-β peptide (1 µM) (Chang et al., 2000) (Fig. 4). Taken together, these findings indicate that the H3R-mediated antiexocytotic effect derives from the Gβ-dependent inhibition of ICa and not from a β-induced inhibition of adenylyl cyclase.
Activation of protein kinase C (PKC) upon application of phorbol esters is known to increase ICa (Zamponi et al., 1997), an action negatively modulated by Gβ subunits (Herlitze et al., 1996; Ikeda, 1996). Accordingly, we next assessed whether H3R stimulation would attenuate PKC-activated ICa and associated increase in dopamine exocytosis via a Gβ-mediated effect. Incubation of PC12-H3 cells with PMA (300 nM) enhanced peak ICa by 30% (compare Figs. 5A and 6A). In the presence of imetit (100 nM), PMA-stimulated ICa was reduced by 45%, and this response was antagonized by clobenpropit (50 nM) (Fig. 6A). PMA also elicited a concentration-dependent increase in dopamine release: in the 10 nM to 1 µM PMA concentration range, dopamine release was increased 2 to 3-fold above control in both PC12-H3 and PC12-H3/β-ARK1 cells (Fig. 7, A and B). In the presence of imetit (100 nM), the concentration-response curve for PMA was shifted to the right by two orders of magnitude in PC12-H3 cells (Fig. 7A). Incubation with clobenpropit (50 nM) antagonized the effect of imetit (Fig. 7C).
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30% (Fig. 6B). It is noteworthy that activation of H3R with imetit in PC12-H3/β-ARK1 cells failed to attenuate the PMA-stimulated ICa (Fig. 6B). Moreover, in PC12-H3/β-ARK1 cells, H3R activation did not modify PMA-induced dopamine exocytosis (Fig. 7B). Indeed, the PMA concentration-response curve for the promotion of dopamine exocytosis was the same whether in the presence or absence of imetit (Fig. 7B), and the magnitude of the exocytotic response to the 300 nM concentration of PMA did not differ from that recorded in the presence of imetit, either alone or together with clobenpropit (Fig. 7D). Thus, stimulation of PKC in PC12-H3 and PC12-H3/β-ARK1 cells confirmed that a Gβ-dependent inhibition of ICa plays a pivotal role in the antiexocytotic effect of H3R activation.
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subunits, liberated from Gβ trimers upon H3R activation attenuate neuronal ICa and consequent neurotransmitter exocytosis. This establishes a novel mechanism of H3R transduction capable of preventing excessive norepinephrine release, a recognized cardioprotective action (Levi et al., 2007).
In our investigation, we used the PC12 rat pheochromocytoma cell line, which acquires a sympathetic nerve phenotype when treated with NGF (Dichter et al., 1977; Taupenot, 2007). Having stably transfected PC12 cells with human H3R, a PTX-sensitive Gi/o-coupled receptor (see Figs. 2 and 4), we set out to demonstrate (using the whole-cell patch clamp technique) the presence of an ICa possibly responsible for the exocytosis of dopamine, the endogenous transmitter in these cells (Chen and Westfall, 1994). Indeed, by stepping to different test potentials in the -40 to +40 mV range, we identified a current in PC12-H3 cells with typical ICa characteristics (i.e., shape of the I/V curve, its persistence in the absence of Na+ in the bathing solution, and its sensitivity to 0.1 mM CdCl2) that was also inhibited by the L- and N-type Ca2+ channel blockers nifedipine and -CTX, respectively. Moreover, this ICa was amplified by stimulation of adenylyl cyclase with forskolin and by PKC activation with a phorbol ester, responses that are typical of neuronal Ca2+ channel currents (Bean et al., 1984; Yang and Tsien, 1993). It is noteworthy that H3R activation with the selective agonist imetit (Garbarg et al., 1992) markedly reduced the ICa in control and both adenylyl cyclase- or PKC-stimulated conditions, an action inhibited by H3R blockade with the selective antagonist clobenpropit (Van der Goot et al., 1992). These findings reveal that the decrease in intracellular Ca2+, which we had previously described to occur in response to H3R activation (Silver et al., 2002; Seyedi et al., 2005), originates from a reduction in Ca2+ influx via VOCC.
To determine whether the H3R-mediated decrease in ICa is due to inhibition of VOCC by the Gi/o-derived Gβ dimer, we transfected PC12-H3 cells with the Gβ scavenger β-ARK1 (Koch et al., 1994). H3R activation in PC12-H3/β-ARK1 cells failed to inhibit ICa, unequivocally demonstrating the pivotal role played by the Gβ dimer in the H3R-mediated inhibition of VOCC. It is clear that the Gβ dimer-induced VOCC inhibition is also of major importance for the antiexocytotic effects of H3R activation. Indeed, we found that the imetit-induced inhibition of dopamine exocytosis in PC12-H3 cells was prevented not only by a membrane-permeable phosducin-like anti-β peptide (Chang et al., 2000), but also in cells transfected with the Gβ scavenger β-ARK1. Moreover, the Gβ scavenger prevented the H3R-mediated attenuation of dopamine exocytosis elicited by PKC activation with the phorbol ester PMA.
It is interesting to note that whereas nifedipine and -CTX each inhibited ICa, only nifedipine attenuated dopamine release. Thus, although both L- and N-type Ca2+ channels are present in PC12-H3 cells, only the L-type seems to be involved in dopamine exocytosis. In fact, calcium entering through the L-type channel is the likely predominant stimulus for dopamine release in PC12 cells (Avidor et al., 1994; Kanwal et al., 1997). Furthermore, although the Gβ dimer is generally viewed as an N-type Ca2+ channel inhibitor (Herlitze et al., 1996; Ikeda, 1996; Catterall, 2000), a Gβ-induced inhibition of the L-type Ca2+ channel has also been recognized (Ivanina et al., 2000). In contrast to dopamine release in PC12 cells, both L- and N-type Ca2+ channels participate in neurotransmitter release from cardiac sympathetic nerve endings. Indeed, we had found that the H3R agonist imetit synergizes with either nifedipine or -CTX to attenuate norepinephrine exocytosis from cardiac synaptosomes (Seyedi et al., 2005).
Expression of the β-ARK1 polypeptide failed to modify the H3R-mediated attenuation of the forskolin-induced increase in intracellular cAMP and associated dopamine exocytosis (see Fig. 2). Although a β-induced inhibition of adenylyl cyclase has been described in insect ovarian cells (Taussig et al., 1993), our findings clearly indicate that the Gβ dimer liberated upon H3R activation is not responsible for the reduction of adenylyl cyclase activity in differentiated PC12-H3 or PC12-H3/β-ARK1 cells. Therefore, a direct β-induced inhibition of ICa at the VOCC level is likely to play a key role in the H3R-mediated antiexocytotic effect. The Gβ dimer is also liberated upon prostaglandin E2-induced activation of EP3R, another Gi/o-coupled receptor with antiexocytotic properties. Prostaglandin E2 formation is the final step of the mitogen-activated protein kinase signaling cascade initiated by H3R activation, culminating in the attenuation of norepinephrine exocytosis from cardiac sympathetic nerve endings (Levi et al., 2007). This substantiates the present findings on the pivotal role of the Gβ dimer as an inhibitor of ICa and consequent exocytosis.
We had previously reported that the antiexocytotic effect of H3R activation is associated with a decrease in intraneuronal Ca2+ concentration in SH-SY5Y human neuroblastoma cells stably transfected with H3R (Silver et al., 2002). We hypothesized that the decrease in [Ca2+]i was due to a Gi-induced inhibition of adenylyl cyclase, ultimately resulting in a decreased PKA-induced phosphorylation of VOCC (Seyedi et al., 2005). The novelty of the present findings is that a direct Gβ-induced inhibition of VOCC, resulting in an attenuation of ICa, plays a pivotal role in the H3R-mediated decrease in [Ca2+]i and associated antiexocytotic effects. The identification of this final step in the H3R transduction cascade has broad implications in the development of new therapeutic strategies in cardiovascular diseases characterized by hyperadrenergic activity, such as myocardial ischemia and congestive heart failure.
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Footnotes |
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C.M. and R.E. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: H3R, histamine H3-receptors; [Ca2+]i, intraneuronal Ca2+; VOCC, voltage-operated Ca2+-channels; PKA, protein kinase A; ICa, Ca2+ current; NGF, nerve-growth factor; β-ARK1, β-adrenergic receptor kinase 1; PCR, polymerase chain reaction; bp, base pairs; TBS, Tris-buffered saline; IMBX, 3-isobutyl-1-methylxanthine; CBP, clobenpropit; PTX, pertussis toxin; PMA, phorbol 12-myristate 13-acetate; act, time constant of activation; -CTX, -conotoxin GVIA; ANOVA, analysis of variance; PKC, protein kinase C.
Address correspondence to: Dr. Roberto Levi, Room LC419, Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065-4896. E-mail address: rlevi{at}med.cornell.edu
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