RESUMO
TASK channels belong to the family of K(+) channels with 4 transmembrane segments and 2 pore domains (4TM/2P) per subunit. These channels have been related to apoptosis in cerebellar granule neurons (CGN), as well as cancer in other tissues. TASK current is regulated by hormones, neurotransmitters, anesthetics and divalent cations, which are not selective. Recently, there has been found some organic compounds that inhibit TASK current selectively. In order to find other modulators, we report here a group of five dihydropyrrolo[2,1-a]isoquinolines (DPIs), four of them with putative anticancer activity, that were evaluated on TASK-1 and TASK-3 channels. The compounds 1, 2 and 3 showed IC50 < 320 µM on TASK-1 and TASK-3, intermediate activity on TASK-1/TASK-3 heterodimer, moderate effect over hslo and TREK-1 (500 µM), and practically not inhibition on Shaker-IR, herg and IRK2.1 potassium channels, when they were expressed heterologously in Xenopus laevis oocytes. In rat CGN, 500 µM of these three compounds induced a decrement by >39% of the TASK-carried leak current. Finally, only compound 1 showed significant protection (â¼36%) against apoptotic death of CGN induced by K(+) deprivation. These results suggest that DPI compounds could be potential candidates for designing new selective inhibitors of TASK channels.
Assuntos
Isoquinolinas/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Pirróis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Isoquinolinas/química , Camundongos , Estrutura Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Pirróis/química , Ratos , Ratos Wistar , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevisRESUMO
Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca(v)1 and Ca(v)2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca(v)3 family) is less documented. We have studied the blocking effect of Cd2+, Co2+ and Ni2+ on T-currents expressed by human Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca2+ (2 mM: ) currents from HEK-293 cells stably expressing recombinant T-type channels. Cd2+ and Co2+ block was 2- to 3-fold more potent for Ca(v)3.2 channels (EC50 = 65 and 122 microM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co2+ and Ni2+ shift the voltage dependence of Ca(v)3.1 and Ca(v)3.3 channels activation to more positive potentials. Interestingly, block of those two Ca(v)3 channels by Co2+ and Ni2+ was drastically increased at extreme negative voltages; in contrast, block due to Cd2+ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd2+ on Ca(v)3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.
Assuntos
Cádmio/fisiologia , Canais de Cálcio Tipo T/metabolismo , Cobalto/fisiologia , Cádmio/química , Canais de Cálcio Tipo T/biossíntese , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/fisiologia , Linhagem Celular , Cobalto/química , Humanos , Cinética , Potenciais da Membrana/fisiologia , Níquel/química , Níquel/fisiologia , Técnicas de Patch-ClampRESUMO
Peptide secretion from rat melanotropes is tonically inhibited by a dopaminergic synaptic input that develops after birth and acts through D2 dopamine receptors. In this study, whole-cell Na(+) currents were recorded from melanotropes that were isolated from rat pituitary intermediate lobes at postnatal days 1-20 (P1-P20) and maintained in culture for 5-24 h. Coincident with the development of innervation, melanotropes exhibited a progressive decrease in peak Na(+) current density from P3 to P14. The decrease involved a 50% reduction in maximal Na(+) conductance with no detectable changes in channel gating. Subcutaneous injections of the D2 antagonist sulpiride, applied from P11 to P13, restored melanotrope Na(+) channel activity to pre-innervation levels. Thus, the activation of D2 receptors by the dopaminergic input reduces the functional expression of Na(+) channels in melanotropes.
Assuntos
Animais Recém-Nascidos/metabolismo , Dopamina/fisiologia , Hormônios Estimuladores de Melanócitos/metabolismo , Hipófise/fisiologia , Canais de Sódio/fisiologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos/genética , Células Cultivadas , Condutividade Elétrica , Hipófise/crescimento & desenvolvimento , Ratos , Ratos WistarRESUMO
1. We have examined the voltage-dependent Ca2+ channel activity of rat melanotrophs during the early postnatal period. The cells were dissociated from pituitary intermediate lobes, kept in culture for 5-24 h and then subjected to whole-cell patch-clamp experiments. 2. Like their adult counterparts, neonatal melanotrophs were able to generate Na+ currents, K+ currents and Ca2+ currents in response to membrane depolarization. Ca2+ currents were carried by both low- and high-threshold Ca2+ channels. 3. High-threshold Ca2+ current density decreased sharply between postnatal day 4 (P4) and P12. This period coincides with the onset of dopaminergic innervation within the intermediate lobe. Accordingly, the developmental decrease in Ca2+ current density was largely reversed by chronic in vivo treatment with sulpiride, a dopamine D2 receptor antagonist. 4. Prolonging the time in culture from 5 h to 8 days did not significantly alter the Ca2+ channel activity of P3 melanotrophs, whereas the high-threshold Ca2+ current in previously innervated (P14) melanotrophs stayed small for the first 24 h and then increased 3-fold during the subsequent 4-5 days. This increase required RNA and protein synthesis and was prevented by adding D2 agonists to the culture medium. 5. These results provide evidence for a postnatal suppression of high-threshold Ca2+ current expression in pituitary melanotrophs mediated by presynaptic dopamine neurons through D2 dopamine receptors.
Assuntos
Canais de Cálcio/metabolismo , Hipófise/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Animais Recém-Nascidos , Bromocriptina/farmacologia , Canais de Cálcio/efeitos dos fármacos , Cicloeximida/farmacologia , Antagonistas de Dopamina/farmacologia , Estimulação Elétrica , Eletrofisiologia , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Receptores de Dopamina D2/agonistas , Sulpirida/farmacologiaRESUMO
In rat pituitary GH3 cells, epidermal growth factor (EGF) and insulin stimulate prolactin production, whereas glucocorticoids exert the opposite effect. In the present study, GH3 cells were subjected to whole-cell patch clamp to assess the chronic actions of such regulatory factors on voltage-dependent calcium currents. Before the electrical recording, cells were grown 5-6 d either under standard conditions or in the presence of 5 nM EGF, 100 nM insulin, 1 microM dexamethasone or 5 microM cortisol. EGF induced a twofold selective increase in high-threshold calcium current density. Insulin and glucocorticoids, on the other hand, specifically regulated low-threshold Ca channels. Current density through these channels increased by 70% in insulin-treated cells, and decreased by 50% in cells exposed to dexamethasone or cortisol. Other Ca channel properties investigated (conductance-voltage curves, deactivation rates, time course and voltage dependence of low-threshold current inactivation) were unaffected by the chemical messengers. The alterations in current density persisted for many hours after removing the regulatory factors from the culture medium. In fact, the stimulatory action of EGF on high-threshold current lasted > 3 d. The results suggest that the control of prolactin production by the factors tested involves regulation of the surface density of functional Ca channels in the plasma membrane.