RESUMO
We tested whether genetic deletion of Cav3.2 T-type Ca2+ channels abolishes hydrogen sulfide (H2S)-mediated pain signals in mice. In Cav3.2-expressing HEK293 cells, Na2S, an H2S donor, at 100 µM clearly increased Ba2+ currents, as assessed by whole-cell patch-clamp recordings. In wild-type C57BL/6 mice, intraplantar and intracolonic administration of Na2S evoked mechanical allodynia and visceral nociceptive behavior, respectively, which were abolished by TTA-A2, a T-type Ca2+ channel blocker. In Cav3.2-knockout mice of a C57BL/6 background, Na2S caused neither somatic allodynia nor colonic nociception. Our study thus provides definitive evidence for an essential role of Cav3.2 in H2S-dependent somatic and colonic pain.
Assuntos
Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Sulfeto de Hidrogênio/farmacologia , Dor Nociceptiva/metabolismo , Transdução de Sinais/efeitos dos fármacos , Dor Visceral/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Canal de Cátion TRPA1/metabolismoRESUMO
The directional migration of epithelial cells is crucial for wound healing. Among integrins, a family of cell adhesion receptors, integrin ß4 has been assumed to be a promigratory factor, in addition to its role in stable adhesion. In turn, Ca(2+) signaling is also a key coordinator of migration. Keratinocytes reportedly express transient receptor potential vanilloid channels (TRPV1); however, the function of these channels as a regulator of intracellular Ca(2+) level in cell migration has remained uncharacterized. In the present study, we investigated the role of TRPV1 in directional migration related to integrin ß4 using a scratch wound assay on a confluent monolayer sheet of murine keratinocytes (Pam212 cells). Double immunofluorescence staining revealed the de novo expression of integrin ß4 and TRPV1 in migrating cells at the wound edge in response to scratch wounding, and both expression levels were almost matched. Epidermal growth factor (EGF) not only promoted keratinocyte migration, but also caused the further up-regulation of both integrin ß4 and TRPV1. In addition, the knockdown of the integrin ß4 or TRPV1 gene significantly impeded wound closure. The TRPV1 agonist capsaicin significantly promoted migration, while a selective TRPV1 antagonist inhibited it. The gene knockdown of TRPV1 inhibited the expression of the integrin ß4 gene and that of ß4 protein in migrating cells. These findings suggest that TRPV1 may stimulate directional migration directly by eliciting a Ca(2+) signal or indirectly via integrin ß4 expression.
Assuntos
Integrina alfa6beta4/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Capsaicina/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cobalto/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Inativação Gênica , Integrina alfa6beta4/genética , Queratinócitos/efeitos dos fármacos , Camundongos , Canais de Cátion TRPV/genética , Regulação para Cima , CicatrizaçãoRESUMO
Hydrogen sulfide (H2S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H2S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Cav3.2 T-type Ca(2+) channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Cav3.2. In the present study, we asked if endogenous and exogenous H2S participates in regulation of the channel functions in Cav3.2-transfected HEK293 (Cav3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Cav3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5mM did not affect T-currents in Cav3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5mM, but not 0.1-0.3mM, increased T-currents in Cav3.2-HEK293 cells. Similarly, Na2S, another H2S donor, at 0.1-0.3mM significantly increased T-currents in the presence, but not absence, of PPG in Cav3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na2S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na2S was higher than NaHS. These results suggest that the function of Cav3.2 T-channels is tonically enhanced by endogenous H2S synthesized by CSE in Cav3.2-HEK293 cells, and that exogenous H2S is capable of enhancing Cav3.2 function when endogenous H2S production by CSE is inhibited. In addition, Na2S is considered a more potent H2S donor than NaHS in vitro as well as in vivo.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Alcinos/farmacologia , Animais , Western Blotting , Cálcio/metabolismo , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular Tumoral , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/farmacologia , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfetos/síntese química , Sulfetos/metabolismo , Sulfetos/farmacologiaRESUMO
Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.
Assuntos
Canais de Cálcio Tipo T , Cistite , Sulfeto de Hidrogênio , Pancreatite , Dor Visceral , Camundongos , Humanos , Animais , Células HEK293 , Canais de Cálcio Tipo T/fisiologia , Sulfetos/farmacologia , Cistite/induzido quimicamente , Sulfeto de Hidrogênio/metabolismoRESUMO
T-type Ca2+ channels (T-channels), particularly Cav3.2 and Cav3.1 isoforms, are promising targets for treating various diseases including intractable pain. Given the potent inhibitory activity of pimozide, an antipsychotic, against T-channels, we conducted structure-activity relationship studies of pimozide derivatives, and identified several compounds including 3a, 3s, and 4 that had potency comparable to that of pimozide in inhibiting T-channels, but little binding affinity to dopamine D2 receptors. The introduction of a phenylbutyl group on the benzoimidazole nuclei of pimozide was considered a key structural modification to reduce the binding affinity to D2 receptors. Those pimozide derivatives potently suppressed T-channel-dependent somatic and visceral pain in mice, without causing any motor dysfunctions attributable to D2 receptor blockade, including catalepsy. The present study thus provides an avenue to develop novel selective T-channel inhibitors available for pain management via the structural modification of existing medicines.
Assuntos
Canais de Cálcio Tipo T , Dor Visceral , Camundongos , Animais , Pimozida/farmacologia , Pimozida/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Dor Visceral/tratamento farmacológico , Dopamina , Bloqueadores dos Canais de Cálcio/química , Receptores Dopaminérgicos/metabolismoRESUMO
ProTx-I peptide, a venom toxin of the tarantula Thrixopelma pruriens, has been reported to interact with voltage-gated ion channels. ProTx-I reduced Ba(2+) currents through recombinant human T-type voltage-gated Ca(2+) channels, Ca(v)3.1 (hCa(v)3.1), with roughly 160-fold more potency than through hCa(v)3.2 channels. Chimeric channel proteins (hCa(v)3.1/S3S4 and hCa(v)3.2/S3S4) were produced by exchanging fourteen amino acids in the hCa(v)3.1 domain IV S3-S4 linker region and the corresponding region of hCa(v)3.2 between each other. The ProTx-I sensitivity was markedly reduced in the hCa(v)3.1/S3S4 chimera as compared to the original hCa(v)3.1 channel, while the hCa(v)3.2/S3S4 chimera exhibited greater ProTx-I sensitivity than the original hCa(v)3.2 channel. These results suggest that the domain IV S3-S4 linker in the hCa(v)3.1 channel may contain residues involved in the interaction of ProTx-I with T-type Ca(2+) channels.
Assuntos
Canais de Cálcio Tipo T/efeitos dos fármacos , Ativação do Canal Iônico , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo T/química , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Venenos de Aranha/farmacologia , Xenopus laevisRESUMO
Since Cav3.2â¯T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293â¯cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (µM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15â¯cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.
Assuntos
Analgésicos não Narcóticos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Flavonoides/farmacologia , Neuralgia/tratamento farmacológico , Dor Visceral/tratamento farmacológico , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/isolamento & purificação , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Modelos Animais de Doenças , Flavonoides/química , Flavonoides/isolamento & purificação , Células HEK293 , Humanos , Humulus , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuralgia/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Distribuição Aleatória , Ratos Wistar , Dor Visceral/metabolismoRESUMO
We have recently demonstrated that intrathecal injection of a selective P/Q-type blocker of the voltage-dependent Ca(2+) channels (VDCCs) significantly inhibited the mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice, its antinociceptive effect being greater than in controls. In this study, to further clarify the underlying mechanism of the STZ-induced hyperalgesia, we investigated the expression level of the VDCC alpha1A and alpha1B subunits in the dorsal root ganglia (DRGs) and the dorsal spinal cord under this hyperalgesia. Real-time PCR analysis showed mRNA expression of alpha1A (P/Q-type), but not alpha1B (N-type), was significantly increased in the DRGs from the STZ-induced diabetic mice. On the other hand, gene expression of both alpha1 subunits was not changed in the dorsal part of the spinal cord. In diabetic DRG neurons, the number of large nerve cells was significantly reduced, whereas small neurons were significantly increased. Immunohistochemical study demonstrated the alpha1A-positive neurons, but not alpha1B-positive neurons, increased significantly greater in diabetic DRGs than in control in all cell size. These results indicate that an alteration in expression of P/Q-type VDCCs, especially in the small and medium-diameter primary afferent fibers, in pain pathways ascending input to the spinal cord may be involved in hypersensitivity in STZ-induced diabetes.
Assuntos
Canais de Cálcio Tipo N/biossíntese , Canais de Cálcio Tipo P/biossíntese , Canais de Cálcio Tipo Q/biossíntese , Diabetes Mellitus Experimental/metabolismo , Gânglios Espinais/metabolismo , Animais , Diabetes Mellitus Experimental/complicações , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
T-type Ca channels (T channels), particularly Cav3.2 among the 3 isoforms, play a role in neuropathic and visceral pain. We thus characterized the effects of RQ-00311651 (RQ), a novel T-channel blocker, in HEK293 cells transfected with human Cav3.1 or Cav3.2 by electrophysiological and fluorescent Ca signaling assays, and also evaluated the antiallodynic/antihyperalgesic activity of RQ in somatic, visceral, and neuropathic pain models in rodents. RQ-00311651 strongly suppressed T currents when tested at holding potentials of -65 â¼ -60 mV, but not -80 mV, in the Cav3.1- or Cav3.2-expressing cells. RQ-00311651 also inhibited high K-induced Ca signaling in those cells. In mice, RQ, administered intraperitoneally (i.p.) at 5 to 20 mg/kg or orally at 20 to 40 mg/kg, significantly suppressed the somatic hyperalgesia and visceral pain-like nociceptive behavior/referred hyperalgesia caused by intraplantar and intracolonic administration of NaHS or Na2S, H2S donors, respectively, which involve the enhanced activity of Cav3.2 channels. RQ-00311651, given i.p. at 5 to 20 mg/kg, exhibited antiallodynic or antihyperalgesic activity in rats with spinal nerve injury-induced neuropathy or in rats and mice with paclitaxel-induced neuropathy. Oral and i.p. RQ at 10 to 20 mg/kg also suppressed the visceral nociceptive behavior and/or referred hyperalgesia accompanying cerulein-induced acute pancreatitis and cyclophosphamide-induced cystitis in mice. The analgesic and antihyperalgesic/antiallodynic doses of oral and i.p. RQ did not significantly affect the locomotor activity and motor coordination. Together, RQ is considered a state-dependent blocker of Cav3.1/Cav3.2 T channels and may serve as an orally available analgesic for treatment of neuropathic and inflammatory pain including distinct visceral pain with minimum central side effects.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Nociceptividade/efeitos dos fármacos , Dor Visceral/tratamento farmacológico , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Hiperalgesia/induzido quimicamente , Masculino , Camundongos , Neuralgia/induzido quimicamente , Paclitaxel , Ratos , Ratos Wistar , Dor Visceral/induzido quimicamenteRESUMO
Intrathecally (i.t.) administered ruthenium red (40, 80, 160 ng) dose-dependently inhibited formalin-induced nociceptive response. Ruthenium red caused a significant inhibition of pain-related behavioral responses induced by i.t. capsaicin but not by i.t. substance P. These results suggest that ruthenium red produces analgesia by inhibiting the release of neuropeptides in the spinal cord.
Assuntos
Analgésicos/farmacologia , Rutênio Vermelho/farmacologia , Analgésicos/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Capsaicina , Formaldeído , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos , Nociceptores/efeitos dos fármacos , Dor/induzido quimicamente , Dor/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Rutênio Vermelho/administração & dosagem , Substância PRESUMO
A modified formalin test in mice was investigated. The pain response curve induced by 0.5% formalin was biphasic, having 2 peaks, from 0 to 5 min (first phase) and from 15 to 20 min (second phase). A low concentration of formalin was used, allowing the effects of weak analgesics to be detected. Centrally acting drugs such as narcotics inhibited both phases equally. Peripherally acting drugs such as aspirin, oxyphenbutazone, hydrocortisone and dexamethasone only inhibited the second phase. Aminopyrine and mefenamic acid which acted on both central and peripheral sites inhibited both phases, but the second phase was inhibited by lower doses. Thus, this method enables one to easily distinguish the site of action of analgesics. Furthermore, pain response in the first phase was inhibited by capsaicin-treated desensitization and Des-Arg9-(Leu8)-bradykinin (bradykinin inhibitor). The second phase was inhibited by compound 48/80 pretreatment, indomethacin and bradykinin inhibitor. Therefore, it is suggested that substance P and bradykinin participate in the manifestation of the first phase response, and histamine, serotonin, prostaglandin and bradykinin are involved in the second phase. These results indicate that the first and second phase responses induced by formalin have distinct characteristic properties, and it is a very useful method for examining pain, nociception and its modulation by pharmacological or other means.
Assuntos
Formaldeído , Medição da Dor/métodos , Dor/fisiopatologia , Analgésicos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Bradicinina/antagonistas & inibidores , Bromelaínas/farmacologia , Capsaicina/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Indometacina/farmacologia , Masculino , Camundongos , Morfina/farmacologia , Fibras Nervosas/fisiologia , Nervo Isquiático/efeitos dos fármacos , Substância P/fisiologia , Fatores de Tempo , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
1. The effects of intrathecal (i.t.) administration of N-, P/Q- or L-type voltage-dependent Ca(2+)-channel blockers were tested in two pain models involving bradykinin (BK)- and alpha,beta-methylene ATP (alpha,beta meATP)-induced activation of primary afferent neurons in mice. 2. The nociceptive response (amount of time spent licking and biting the hindpaw) induced by intraplantar injection of BK (500 pmol mouse(-1)) was significantly attenuated by both omega-conotoxin GVIA (N-type blocker) and calciseptine (L-type) but not by omega-agatoxin IVA (P/Q-type). 3. The nociceptive response induced in a similar way by alpha,beta meATP (100 nmol) was significantly inhibited by both the above N- and P/Q-type Ca(2+)-channel blockers but not by the L-type blocker. 4. The nociceptive responses elicited by BK and alpha,beta meATP were dose-dependently inhibited by a tachykinin-NK1-receptor antagonist (L-703,606) and an N-methyl-D-aspartate (NMDA)-receptor antagonist (D-AP5), respectively. 5. Intrathecal administration of substance P (SP) (1.8 nmol) or NMDA (350 pmol) elicited algesic responses, such as licking, biting and scratching of the hindquarters. The SP-induced algesic behaviour was significantly inhibited by the L-type blocker but not by the N-type. The NMDA-induced response was not affected by either the N- or the P/Q-type blocker. 6. These findings suggest that BK and ATP most likely excite different types of sensory neurons in the periphery and that within the spinal cord the former stimulates peptidergic transmission regulated by presynaptic N- and postsynaptic L-type Ca(2+) channels, while the latter stimulates glutamatergic transmission regulated by presynaptic N- and P/Q-type channels.
Assuntos
Trifosfato de Adenosina/farmacologia , Bradicinina/farmacologia , Canais de Cálcio/fisiologia , Dor/fisiopatologia , Medula Espinal/fisiologia , Transmissão Sináptica/fisiologia , Trifosfato de Adenosina/análogos & derivados , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Nociceptores/fisiologia , Limiar da Dor/fisiologia , Medula Espinal/efeitos dos fármacosRESUMO
Our aim was to investigate the possible involvement of spinal voltage-dependent Ca(2+) channels (VDCCs) in vincristine-induced hyperalgesia and also to characterize this hyperalgesic state in the spinal cord. Mice receiving vincristine displayed significantly lower mechanical nociceptive thresholds than controls. Intrathecal omega-conotoxin GVIA (an N-type blocker) produced dose-dependent inhibition of the mechanical nociception, its antinociceptive effect being greater in vincristine-treated mice than in controls. The antinociception of omega-agatoxin IVA (a P/Q-type blocker) and calciseptine (an L-type blocker) were both slightly, but not significantly greater in vincristine-treated mice. An N-methyl-D-aspartate-receptor antagonist but not a tachykinin-NK1-receptor antagonist produced greater antinociception in vincristine-treated mice. These results suggest that vincristine-induced hyperalgesia involves a sensitization of the spinal processing of mechanical sensory information via a mechanism involving N-type but not P/Q- or L-type VDCCs. A spinal glutamatergic pathway also appears to be involved in this hyperalgesia.
Assuntos
Hiperalgesia/fisiopatologia , Medula Espinal/fisiopatologia , Valina/análogos & derivados , Vincristina/toxicidade , Animais , Antineoplásicos Fitogênicos/toxicidade , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Relação Dose-Resposta a Droga , Venenos Elapídicos/farmacologia , Hiperalgesia/induzido quimicamente , Masculino , Camundongos , Antagonistas dos Receptores de Neurocinina-1 , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Quinuclidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Fatores de Tempo , Valina/farmacologia , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxinas/farmacologiaRESUMO
We studied the antinociceptive effects induced at the spinal level by N-, P/Q- and L-type voltage-dependent Ca2+-channel (VDCC) blockers given alone or in combination with morphine, the test responses being the algesic ones induced by acute thermal and mechanical stimuli. When given alone, intrathecal omega-agatoxin IVA (P/Q-type blocker) produced a potent dose-dependent inhibition in the tail-flick and tail-pressure over the dose range 0.33-33 pmol/mouse. Omega-conotoxin GVIA (N-type blocker) also produced dose-dependent inhibitions, but its antinociception against thermal stimuli was weaker than against mechanical stimuli. Calciseptine (L-type blocker) slightly reduced both nociceptive responses, but only at 33 pmol. At their subthreshold doses, intrathecal omega-agatoxin IVA, omega-conotoxin GVIA and calciseptine each significantly enhanced morphine analgesia in the tail-flick and tail-pressure tests, the rank order of potencies being N-> or =P/Q->L-type. These results indicate that combining a low-dose VDCC blocker, especially the N- or P/Q-type, with morphine may be a very useful way of minimizing the dose of morphine and may reduce side effects.
Assuntos
Analgésicos Opioides/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo N/efeitos dos fármacos , Canais de Cálcio Tipo P/efeitos dos fármacos , Canais de Cálcio Tipo Q/efeitos dos fármacos , Morfina/farmacologia , Medula Espinal/metabolismo , Animais , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Temperatura Alta , Injeções Espinhais , Masculino , Camundongos , Medição da Dor/efeitos dos fármacos , Estimulação Física , Equilíbrio Postural/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
T-type voltage-gated Ca2+ channels have unique electrophysiological properties, suitable for generating Ca2+ oscillations and waves and thus controlling the proliferation of various tumor cells. In the present study, we investigated the role of Cav3.1, a candidate tumor suppressor gene, in neoplastic processes, and compared the differences between Cav3.1 with Cav3.2 channels. While the overexpression of a full-length Cav3.1 clone suppressed cell proliferation, the knockdown of the Cav3.1 gene by siRNA, or treatment with ProTx-I, a relatively selective inhibitor for Cav3.1, promoted the cell proliferation of MCF-7 cells (a human breast adenocarcinoma cell line). Although Cav3.1 and Cav3.2 channels possess comparable biophysical properties and are often co-expressed in various tissues, gene knockdown or the overexpression of Cav3.2 channels exhibited no effect on cell proliferation. Using immunocytochemical co-staining, the Cav3.1 channels were specifically visualized in the plasma membranes of apoptotic cells, identified by Annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays and nuclear condensation. On the contrary, Cav3.2 channels were expressed at the membrane of large portions of cells, with no likely relation to Cav3.1 expression or apoptosis. An apoptosis assay revealed that the overexpression of the Cav3.1 clone caused an increase in the number of apoptotic cells. Furthermore, Cav3.1 knockdown blocked cyclophosphamide-induced apoptosis. These results suggest that Cav3.1 channels may contribute to the repression of tumor proliferation and the promotion of apoptosis mediated via Cav3.1-specific Ca2+ influx.
Assuntos
Apoptose , Canais de Cálcio Tipo T/metabolismo , Proliferação de Células , Neoplasias da Mama , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/genética , Sinalização do Cálcio , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Potenciais da Membrana , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Venenos de Aranha/farmacologiaRESUMO
Several members of the transient receptor potential (TRP)-channel family are expressed in cancer cells. One, cold/menthol-sensitive TRPM8, is reportedly an important player in carcinogenesis in human prostate cancer, although its involvement in oral squamous cell carcinoma (SCC) remains unclear. The present immunohistochemistry and RT-PCR results revealed intense TRPM8 expression in two SCC cell lines, HSC3 and HSC4, derived from the human tongue. Menthol, icilin, and a more specific TRPM8 agonist (WS-12) induced non-specific cation currents, with Ca2+ permeability being greater than that of Na+ or K+. The novel TRPM8 antagonist RQ-00203078 (RQ) profoundly reduced such agonist-induced cation currents. Intracellular Ca2+ imaging revealed that menthol induced both intracellular Ca2+ release and store-operated Ca2+ entry, with RQ inhibiting each effect. To assess the possible pathophysiological role of TRPM8 in oral SCC, we performed motility and invasion assays, and gelatin zymography. Menthol augmented the migration and invasion abilities of both HSC3 and HSC4 cells by potentiating MMP-9 activity. RQ suppressed all of these effects. These results may aid understanding of the pathophysiological implications of TRPM8 channels in the oral SCC cells, support TRP proteins as valuable targets for pharmaceutical intervention, and inform the targeting of oral SCC in which the prognosis is poor.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Movimento Celular/efeitos dos fármacos , Canais de Cátion TRPM/antagonistas & inibidores , Neoplasias da Língua/metabolismo , Anilidas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Metaloproteinase 9 da Matriz/metabolismo , Potenciais da Membrana , Mentol/análogos & derivados , Mentol/farmacologia , Invasividade Neoplásica , Técnicas de Patch-Clamp , Pirimidinonas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Neoplasias da Língua/genética , Neoplasias da Língua/patologiaRESUMO
BACKGROUND AND OBJECTIVE: A genetically diabetic mouse strain (db/db) exhibits severe obesity and a syndrome resembling human non-insulin-dependent diabetes mellitus. Our histological study of submandibular glands revealed that the size and area of the granular convoluted tubules was substantially decreased in db/db mice. We hypothesized that this structural difference reflected a specific alteration in salivary duct function. METHODS: The saliva evoked by pilocarpine was used for the measurement of ion concentrations, and submandibular glands were dissected out for the immunohistochemistry and real-time PCR study. RESULTS: The K(+) concentration of the salivary secretion was higher in db/db than in control m+/m+ mice, while neither saliva volume nor the concentrations of Na(+) or Clâ» differed between these strains. In db/db mice (vs. m+/m+ mice): quantitative PCR analysis revealed an increased mRNA expression of large-conductance Ca²(+)-activated K(+) (maxi-K) channels, immunohistochemistry revealed an increase in the luminal surface expression of the maxi-K channel protein, and a particularly interesting finding was that there was a substantial increase in the salivary tissue-specific splice variant ParSlo. CONCLUSION: These results suggest that in db/db mice, the K(+) content of saliva may be elevated due to an expression of a maxi-K channel variant, which results from a modification of ductal structure. GENERAL SIGNIFICANCE: Our data may shed some light on the mechanism responsible for determining the dynamics of salivary K(+) concentration increased in diabetic patients.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Saliva/química , Glândula Submandibular/metabolismo , Glândula Submandibular/fisiopatologia , Animais , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saliva/metabolismoRESUMO
The aim of this study was to evaluate the difference in the expression of mRNA of two types of calcium channels between longitudinal and circular muscle layer of rat myometrium during pregnancy. Changes in the expressions of the mRNA encoding L-type (alpha1C) and T-type (alpha1G, alpha1H, and alpha1I) calcium channels in longitudinal and circular smooth muscle cells of the rat myometrium were examined using a comparative kinetic RT/PCR method. During the course of pregnancy, alpha1C mRNA expression showed an N-shaped change in longitudinal muscle, but simply increased after mid-pregnancy in circular muscles. The mRNAs for alpha1G and alpha1H, but not that for alpha1I, were expressed in both longitudinal and circular smooth muscle. In longitudinal muscle, the change in alpha1H mRNA was similar to that in alpha1C mRNA during gestation, but the expression of alpha1G mRNA changed significantly only at term (day 22). In circular muscle, alpha1H mRNA expression was stable at any stage during pregnancy, but alpha1G mRNA significantly increased on day 15 and at term. No relationship was observed between voltage-dependent calcium-channel mRNA expressions and either proliferation or hypertrophy of circular muscle during pregnancy. These results show (a) that during pregnancy, the expression levels of L-type channels change dynamically, and it may contribute directly to the regulation of cell excitability, and (b) that the T-subtype that increases during pregnancy differs between longitudinal and circular muscle cells, although their functions remain unclear.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/metabolismo , Miócitos de Músculo Liso/metabolismo , Miométrio/metabolismo , Gravidez/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo T/genética , Feminino , Expressão Gênica , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Electrophysiological characteristics were compared among four cloned human alpha1H isoforms transcripted by alternative splicings of exons 25B and 26 [Delta25B/+26 (native form; alpha1H-a), Delta25B/Delta?6 (alpha1H-b), +25B/Delta26, and +25B/+26] in the intracellular loop between domains III and IV (III-IV linker) of a human T-type calcium channel (Ca(v)3.2). The native isoform Delta25B/+26 predominated in ovary and non-pregnant uterus, while isoform Delta25B/Delta26 (alpha1H-b) predominated in pregnant uterus and testis. Expressions of the newly identified +25B/Delta26 and +25B/+26 isoforms were greater in the uterus at gestation than in the non-pregnant uterus. When expressed in Xenopus laevis oocytes, all isoforms produced transient inward currents with low voltage-dependent activation and inactivation characterized in typical T-type Ca2+ currents. Each isoform possessing exon 25B (+25B/?Delta26 or +25B/+26) showed current activation and inactivation at a more negative membrane potential than the respective isoform (Delta25B/Delta26 or Delta25B/+26) lacking it. Moreover, the current activation and inactivation rates were faster for the two isoforms possessing exon 25B than for the respective isoforms lacking it. By itself, exon 26 seemed not to affect any electrophysiological characteristics. Increasing the net positive charge (relative to the native form), as occurred in isoforms Delta25B/Delta26, +25B/Delta26, and +25B/+26, caused recovery from short-term inactivation to become faster. Our results show that molecular-structure variations within the III-IV linker influence the voltage-dependence and kinetics of both activation and inactivation. Although the role of T-type Ca2+ channels in uterine tissue remains unknown, changes in the uterine expression of these alpha1H isoforms may influence physiological functions during pregnancy.