Arg-vasopressin facilitates calcium channel currents in osteoblasts.

The hypothalamic nonapeptide and neurohypophyseal hormone arg-vasopressin (AVP), also known as antidiuretic hormone, is best known for its effects on water reabsorption in kidney. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of AVP on VDCC currents in osteoblasts using a patch-clamp recording method. An application of 1µM AVP facilitated VDCC currents in osteoblasts. To our knowledge, the data presented here demonstrate for the first time that AVP facilitates VDCCs in osteoblasts.

Adrenaline facilitates calcium channel currents in osteoblasts.

Adrenaline (Adr) is known to directly or indirectly modulate bone cell activity under physiological and pathological conditions. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of Adr on VDCC currents in osteoblasts using a patch-clamp recording method. Application of 1 mM Adr facilitated VDCC currents in a concentration-dependent manner. Pre-treatment with b receptor antagonist propranolol blocked Adr-induced facilitation of VDCC currents carried by Ba(2+) (IBa). These results indicate that Adr-induced facilitation of IBa was mediated by b receptors in MC3T3-E1 osteoblast-like cells. To our knowledge, the data presented here demonstrate for the first time that Adr facilitates VDCCs in MC3T3-E1 osteoblast-like cells.

Angiotensin II induces modulation of calcium channel currents in osteoblasts.

Angiotensin II (Ang II) plays a major role in the maintenance of extracellular fluid volume and blood pressure. In addition to its well-established role in circulatory homeostasis, it has been implicated in the process of bone formation. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of Ang II on VDCC currents in osteoblasts using a patch-clamp recording method. To our knowledge, the data presented here demonstrate for the first time that Ang II facilitates VDCCs in osteoblasts.

Neuropeptide Y modulates calcium channels in hamster submandibular ganglion neurons.

It is established that neuropeptide Y (NPY) is a transmitter of parasympathetic secretory impulses in submandibular gland. The neuropeptides substance P, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are likely mediators of secretory parasympathetic responses of the gland. Previously, we have shown that substance P, VIP and CGRP modulate voltage-dependent Ca(2+) channels (VDCCs) in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to characterize the effect of NPY on VDCCs current using Ba(2+) (I(Ba)) in SMG neurons. Application of NPY caused both facilitation and inhibition of L-type and N/P/Q-type I(Ba), respectively. Intracellular dialysis of the Gα(s)-protein antibody attenuated the NPY-induced facilitation of I(Ba). The adenylate cyclase (AC) inhibitor, as well as protein kinase A (PKA) inhibitor attenuated the NPY-induced facilitation of I(Ba). Intracellular dialysis of the Gα(i)-protein antibody attenuated the NPY-induced inhibition of I(Ba). Application of a strong depolarizing voltage prepulse attenuated the NPY-induced inhibition of I(Ba). These results indicate that NPY facilitates L-type VDCCs via Gα(s)-protein involving AC and PKA. On the other hand, NPY also inhibits N/P/Q-type VDCCs via Gα(i)-protein ßγ subunits in the SMG neurons.

Prepulse facilitation of calcium channel current in osteoblasts.

Osteoblasts play a major role in bone formation. Osteoblasts employ intracellular Ca(2+) as a second messenger modulating hormonal responses and a cofactor for bone mineralization. Voltage-dependent Ca(2+) channels (VDCCs) are most commonly present in excitable cell membranes. They are also present at lower levels even in most nonexcitable cells too. In both types of cell, they mediate the influx of Ca(2+) in response to membrane depolarization. Prepulse facilitation is a phenomenon in which a long and strong depolarizing pulse induces a form of VDCC that exhibits an increased opening probability. We believe this to be the first study to demonstrate that strong depolarization prepulses both increase and decrease VDCCs in osteoblasts.

Chronic bradykinin treatment alters 1α,25-dihydroxyvitamin D3-induced calcium current modulation in pre-osteoblasts.

Bradykinin (BK) is involved in bone resorption in chronic inflammatory diseases. During bone formation, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays an important role in the regulation of Ca2+. In osteoblasts, 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through voltage-sensitive Ca2+ channels (VSCCs). Voltage sensitive Ca2+ channels serve as crucial mediators of membrane excitability and many Ca2+-dependent functions, including bone growth, regulation of proliferation, enzyme activity and gene expression. The purpose of this study was to investigate the effects of BK and 1,25(OH)2D3 on VSCC currents carried by Ba2+ (IBa). Application of 1,25(OH)2D3 facilitated IBa in a voltage-dependent manner. Pretreatment with SQ22536 (an adenylate cyclase inhibitor) attenuated 1,25(OH)2D3-induced facilitation of IBa. Bradykinin and BK1 receptor agonist [Lys-des-Arg9]-BK also facilitated IBa. After 24 h or 7 days exposure to BK, that is, under chronic inflammatory conditions, application of 1,25(OH)2D3 inhibited IBa. In addition, pretreatment with PD98,059, a mitogen-activated protein kinase (MAPK) tyrosine kinase inhibitor, attenuated 1,25(OH)2D3-induced inhibition of IBa. These results indicate that, under normal conditions, 1,25(OH)2D3 acts with adenylate cyclase to facilitate VSCCs, whereas under chronic inflammatory conditions it acts with MAPK to inhibit VSCCs in pre-osteoblasts.

Adrenomedullin facilitates calcium channel currents in osteoblasts.

Osteoblasts play a major role in bone formation. Osteoblasts employ intracellular Ca(2+) as a second messenger to modulate hormonal responses and a cofactor for bone mineralization. Adrenomedullin (ADM) promotes osteoblast growth and proliferation, inducing an increase in bone mass. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. Voltage-dependent Ca(2+) channels serve as crucial mediators of many Ca(2+)-dependent functions, including growth of bone and regulation of proliferation. The purpose of this study was to investigate the effects of ADM on VDCC currents in osteoblasts using a patch-clamp recording method. To our knowledge, the data presented here demonstrate for the first time that ADM facilitates VDCCs in osteoblasts.

Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current in submandibular ganglion.

OBJECTIVE: The control of saliva secretion is mainly under parasympathetic control. The submandibular ganglion (SMG) is a parasympathetic ganglion which receives inputs from preganglionic cholinergic neurons, and innervates the submandibular salivary gland to control saliva secretion. The aim of this study was to investigate if adrenomedullin (ADM) and/or calcitonin gene-related peptide (CGRP) modulate voltage-dependent calcium channel (VDCCs) current (I(Ca)) in SMG. DESIGN: The profile of CGRP and ADM actions in SMG was studied using the whole-cell configuration of the patch-clamp technique. RESULTS: Both ADM and CGRP facilitated I(Ca). These facilitations were attenuated by intracellular dialysis of the anti-Gα(s)-protein and pretreatment of SQ22536 (an adenylate cyclase inhibitor). CONCLUSIONS: ADM and CGRP facilitates VDCCs mediated by Gα(s)-protein and adenylate cyclase in SMG.

1α,25-dihydroxyvitamin D3 rapidly modulates Ca(2+) influx in osteoblasts mediated by Ca(2+) channels.

The biologically active form of vitamin D, 1α,25-dihydroxy vitamin D3 (VD), regulates the synthesis of the bone Ca-binding proteins osteocalcin and osteopontin. The actions of VD are mediated through the vitamin D receptor (VDR). Liganded VDR heterodimerizes with the retinoid X receptor and interacts with a vitamin D response element (VDRE). Recently, it has been demonstrated that vitamin D responses elicited in osteoblasts can be rapid as well as long-term. The purpose of this study was to elucidate the mechanism of Ca(2+) signaling of VD in osteoblasts using intracellular Ca(2+) ([Ca(2+)]i) measurements. A rapid VD (10 nM)-induced increase in [Ca(2+)]i was observed within 40 sec. This increase, however, was negated with application of Ca(2+)-free Krebs' solution. These results indicate that VD induces an increase in [Ca(2+)]i from extracellular Ca(2+) in osteoblasts.

Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current by different signal pathways in nucleus tractus solitarius.

Calcitonin gene-related peptides (CGRP) and adrenomedullin (ADM) belong to the calcitonin family of peptides and are structurally related. Both peptides are found in the neurons of the CNS and play a role in many neuronal functions, including the control of blood pressure. The nucleus tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Recently, hypotension and bradycardia were observed after CGRP and ADM injection in the NTS. Voltage-dependent Ca(2+) channels (VDCCs) serve as crucial mediators of membrane excitability and Ca(2+)-dependent functions, such as neurotransmitter release, enzyme activity, and gene expression. The purpose of this study is to investigate the effects of CGRP and ADM on VDCC currents (I(Ca)) carried by Ba(2+) (I(Ba)) in the NTS, using patch-clamp recording methods. Application of CGRP and ADM caused facilitation of I(Ba) in a concentration-dependent manner. Intracellular dialysis of the anti-Galpha(s)-protein antibody attenuated CGRP-induced facilitation of I(Ba). Intracellular dialysis of the anti-Galpha(i)-protein antibody attenuated ADM-induced facilitation of I(Ba). Pretreatment with SQ22536 (an adenylate cyclase inhibitor) and intracellular dialysis of PKI(5-24) (a protein kinase A inhibitor) attenuated CGRP-induced facilitation of I(Ba). In contrast, pretreatment with PD98,059 (a mitogen-activated protein kinas inhibitor) attenuated ADM-induced facilitation of I(Ba). Mainly L-type VDCCs were facilitated by both CGRP and ADM. These results indicate that CGRP facilitates L-type VDCCs via Galpha(s)-protein involving adenylate cyclase and protein kinase A. In contrast, ADM facilitates L-type VDCCs via Galpha(i)-protein involving mitogen-activated protein kinase in the NTS.

CbpA: a polarly localized novel cyclic AMP-binding protein in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, cyclic AMP (cAMP) signaling regulates the transcription of hundreds of genes encoding diverse virulence factors, including the type II secretion system (T2SS) and type III secretion system (T3SS) and their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent transcriptional regulator that is homologous to the Escherichia coli catabolite repressor protein, is thought to be the major cAMP-binding protein that regulates these important virulence determinants. Using a bioinformatic approach, we have identified a gene (PA4704) encoding an additional putative cAMP-binding protein in P. aeruginosa PAO1, which we herein refer to as CbpA, for cAMP-binding protein A. Structural modeling predicts that CbpA is composed of a C-terminal cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. We show that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using in vitro trypsin protection assays, we demonstrate that CbpA undergoes a conformational change upon cAMP binding. Reporter gene assays and electrophoresis mobility shift assays defined the cbpA promoter and a Vfr-binding site that are necessary for Vfr-dependent transcription. Although CbpA is highly regulated by Vfr, deletion of cbpA did not affect known Vfr-dependent functions, including the T2SS, the T3SS, flagellum- or TFP-dependent motility, virulence in a mouse model of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green fluorescent protein was found to be localized to the flagellated old cell pole in a cAMP-dependent manner. These results suggest that polar localization of CbpA may be important for its function.

Identification of the electron transfer flavoprotein as an upregulated enzyme in the benzoate utilization of Desulfotignum balticum.

Desulfotignum balticum utilizes benzoate coupled to sulfate reduction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was conducted to detect proteins that increased more after growth on benzoate than on butyrate. A comparison of proteins on 2D gels showed that at least six proteins were expressed. The N-terminal sequences of three proteins exhibited significant identities with the alpha and beta subunits of electron transfer flavoprotein (ETF) from anaerobic aromatic-degraders. By sequence analysis of the fosmid clone insert (37,590 bp) containing the genes encoding the ETF subunits, we identified three genes, whose deduced amino acid sequences showed 58%, 74%, and 62% identity with those of Gmet_2267 (Fe-S oxidoreductase), Gmet_2266 (ETF beta subunit), and Gmet_2265 (ETF alpha subunit) respectively, which exist within the 300-kb genomic island of aromatic-degradation genes from Geobacter metallireducens GS-15. The genes encoding ETF subunits found in this study were upregulated in benzoate utilization.

Nerve growth factor and brain-derived neurotrophic factor attenuate angiotensin-II-induced facilitation of calcium channels in acutely dissociated nucleus tractus solitarii neurons of the rat.

OBJECTIVE: Neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), promote neuronal development and neuronal survival, but their mechanisms remain controversial. This study aimed to investigate the hypothesis that NGF and BDNF interfere with angiotensin-II- and glutamate-induced facilitation of voltage-dependent Ca(2+) channels (VDCCs) in nucleus tractus solitarius (NTS) neurons. DESIGN: The profile of NGF and BDNF actions in acutely dissociated rat NTS was studied using the whole-cell configuration of the patch-clamp technique. RESULTS: Pretreatment with NGF and BDNF attenuated angiotensin-II-induced facilitation of VDCCs, but did not attenuate glutamate-induced facilitation of the L-type VDCC current in NTS neurons. NGF-induced attenuation was antagonised by pretreatment with a tyrosine kinase A (TrkA) receptor antagonist K-252a. CONCLUSIONS: NGF attenuated angiotensin-II-induced facilitation of L-type VDCCs mediated by TrkA receptors in NTS neurons.

Galanin inhibits calcium channels via Galpha(i)-protein mediated by GalR1 in rat nucleus tractus solitarius.

Galanin (GAL), a 29-amino-acid neuropeptide, is involved in various neuronal functions, including the regulation of food intake, hormone secretion and central cardiovascular regulation. The nucleus tractus solitarius (NTS) is known to plays a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca(2+)-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of GAL on VDCCs currents (ICa) carried by Ba2+ (IBa) in the NTS using patch-clamp recording methods. An application of M617 (GalR1 specific agonist), AR-M961 (GAL receptor GalR 1/2 agonist) and GAL caused inhibition of N- and P/Q-types I(Ba). M617, GAL, and AR-M961 caused inhibition of I(Ba) in a concentration-dependent manner, with IC50s of 678 nM, 325 nM and 573 nM, respectively. This inhibition was relieved, albeit incompletely, by a depolarizing prepulse. Pretreatment with M35 (GalR non-specific antagonist) attenuated the M617-induced inhibition of I(Ba). Intracellular dialysis of the Galpha(i)-protein antibody also attenuated the Gal-induced inhibition of IBa. These results indicate that GAL inhibits N- and P/Q-types VDCCs via Galpha(i)-protein betagamma subunits mediated by GalR1 in NTS.

Transcription factors CysB and SfnR constitute the hierarchical regulatory system for the sulfate starvation response in Pseudomonas putida.

Pseudomonas putida DS1 is able to utilize dimethyl sulfone as a sulfur source. Expression of the sfnFG operon responsible for dimethyl sulfone oxygenation is directly regulated by a sigma(54)-dependent transcriptional activator, SfnR, which is encoded within the sfnECR operon. We investigated the transcription mechanism for the sulfate starvation-induced expression of these sfn operons. Using an in vivo transcription assay and in vitro DNA-binding experiments, we revealed that SfnR negatively regulates the expression of sfnECR by binding to the downstream region of the transcription start point. Additionally, we demonstrated that a LysR-type transcriptional regulator, CysB, directly activates the expression of sfnECR by binding to its upstream region. CysB is a master regulator that controls the sulfate starvation response of the sfn operons, as is the case for the sulfonate utilization genes of Escherichia coli, although CysB(DS1) appeared to differ from that of E. coli CysB in terms of the effect of O-acetylserine on DNA-binding ability. Furthermore, we investigated what effector molecules repress the expression of sfnFG and sfnECR in vivo by using the disruptants of the sulfate assimilatory genes cysNC and cysI. The measurements of mRNA levels of the sfn operons in these gene disruptants suggested that the expression of sfnFG is repressed by sulfate itself while the expression of sfnECR is repressed by the downstream metabolites in the sulfate assimilatory pathway, such as sulfide and cysteine. These results indicate that SfnR plays a role independent of CysB in the sulfate starvation-induced expression of the sfn operons.

Transcriptional regulation of the sulfate-starvation-induced gene sfnA by a sigma54-dependent activator of Pseudomonas putida.

The sigma(54)-dependent transcriptional regulator SfnR is essential for the use of dimethyl sulfone (DMSO(2)) as a sulfur source by Pseudomonas putida DS1. SfnR binds three SfnR-binding sites (sites 1, 2 and 3) within an intergenic region of the divergently transcribed sfnAB and sfnFG gene clusters. The site 1 region, proximal to the sfnF gene, is indispensable for the expression of the sfnFG operon, which encodes components of DMSO(2) monooxygenase. We investigated the transcriptional regulation of the sfnAB operon and possible functions of the sfnA gene. RT-PCR analysis revealed that the sfnAB gene cluster, which is similar to homologues of the acyl-CoA dehydrogenase family, was transcribed as an operon, and its expression was regulated by SfnR under conditions of sulfate starvation. Deletion analyses using lacZ as a reporter demonstrated that the region up to at least -138 bp from the transcription start point of sfnA (containing sites 2 and 3) was necessary for the expression of the sfnAB operon. A growth test of the sfnA-disrupted mutant revealed the possibility that sfnA may be involved in the use of methanethiol as a sulfur source.

The ptsP gene encoding the PTS family protein EI(Ntr) is essential for dimethyl sulfone utilization by Pseudomonas putida.

Many bacteria living in soil have developed the ability to use a wide variety of organosulfur compounds. Pseudomonas putida strain DS1 is able to utilize dimethyl sulfide as a sulfur source via a series of oxidation reactions that sequentially produce dimethyl sulfoxide, dimethyl sulfone (DMSO2), methanesulfonate, and sulfite. To isolate novel genes involved in DMSO2 utilization, a transposon-based mutagenesis of DS1 was performed. Of c. 10,000 strains containing mini-Tn5 inserts, 11 mutants lacked the ability to utilize DMSO2, and their insertion sites were determined. In addition to the cysNC, cysH, and cysM genes involved in sulfate assimilation, the ptsP gene encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family protein EI(Ntr) was identified, which is necessary for DMSO2 utilization. Using quantitative reverse transcriptase-polymerase chain reaction analysis, it was demonstrated that the expression of the sfn genes, necessary for DMSO2 utilization, was impaired in the ptsP disruptant. To the authors' knowledge, this is the first report of a PTS protein that is involved in bacterial assimilation of organosulfur compounds.

Muscarinic M2 receptor inhibition of calcium current in rat nucleus tractus solitarius.

The cholinergic system in the central nervous system plays an important role in higher brain functions, through muscarinic receptors. The nucleus tractus solitarius is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of acetylcholine (Ach) on VDCC currents (I(Ca)) in the nucleus tractus solitarius using patch-clamp recording methods. In 68 out of 99 neurons, an application of ACh caused inhibition of N-type and P/Q-type I(Ba) in a concentration-dependent manner. Pretreatments with AF-DX116 (muscarinic M2 receptor antagonist) attenuated the ACh-induced inhibition of I(Ba). Intracellular dialysis of the Galpha(i)-protein antibody also attenuated the ACh-induced inhibition of I(Ba). These results indicate that ACh inhibits N-type and P/Q-type VDCCs via Gi-protein betagamma subunits mediated by M2 receptors in nucleus tractus solitarius.

Dual effects of neurokinin on calcium channel currents and signal pathways in neonatal rat nucleus tractus solitarius.

Neurokinins, such as substance P (SP), modulate the reflex regulation of cardiovascular and respiratory function in the CNS, particularly in the nucleus tractus solitarius (NTS). There is considerable evidence of the action of SP in the NTS, but the precise effects have not yet been determined. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+ -dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of neurokinins on VDCCs currents (ICa) in the NTS using patch-clamp recording methods. In 142 of 282 neurons, an application of [Sar(9), Met(O(2)11]-substance P (SSP, NK(1) receptor agonist) caused facilitation of L-type I(Ba). Intracellular dialysis of the Galpha(q/11)-protein antibody attenuated the SSP-induced facilitation of I(Ba). In addition, phospholipase C (PLC) inhibitor, protein kinase C (PKC) inhibitor and PKC activator attenuated the SSP-induced the facilitation of I(Ba). In contrast, in 115 of 282 neurons, an application of SSP caused inhibition of N- and P/Q-types I(Ba). Intracellular dialysis of the Gbetagamma-protein antibody attenuated the SSP-induced inhibition of I(Ba). These results indicate that NK(1) receptor facilitates L-type VDCCs via Galpha(q/11)-protein involving PKC in NTS. On the other hand, NK(1) receptor inhibits N- and P/Q-types VDCCs via Galpha(q/11)-protein betagamma subunits in NTS.

Pharmacological characterization of inhibitory effects of postsynaptic opioid and cannabinoid receptors on calcium currents in neonatal rat nucleus tractus solitarius.

1. The profile of opioid and cannabinoid receptors in neurons of the nucleus tractus solitarius (NTS) has been studied using the whole-cell configuration of the patch clamp technique. 2. Experiments with selective agonists and antagonists of opioid, ORL and cannabinoid receptors indicated that mu-opioid, kappa-opioid, ORL-1 and CB1, but not delta-opioid, receptors inhibit VDCCs in NTS. 3. Application of [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; mu-opioid receptor agonist), Orphanin FQ (ORL-1 receptor agonist) and WIN55,122 (CB1 receptor agonist) caused inhibition of I(Ba) in a concentration-dependent manner, with IC50's of 390 nM, 220 nM and 2.2 microM, respectively. 4. Intracellular dialysis of the G(i)-protein antibody attenuated DAMGO-, Orphanin FQ- and WIN55,122-induced inhibition of I(Ba). 5. Both pretreatment with adenylate cyclase inhibitor and intracellular dialysis of the protein kinase A (PKA) inhibitor attenuated WIN55,122-induced inhibition of I(Ba) but not DAMGO- and Orphanin FQ-induced inhibition. 6. Mainly N- and P/Q-type VDCCs were inhibited by both DAMGO and Orphanin FQ, while L-type VDCCs were inhibited by WIN55,122. 7. These results suggest that mu- and kappa-opioid receptors and ORL-1 receptor inhibit N- and P/Q-type VDCCs via G alpha(i)-protein betagamma subunits, whereas CB1 receptors inhibit L-type VDCCs via G alpha(i)-proteins involving PKA in NTS.