RESUMO
Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with increased prevalence of insulin resistance (IR). IR could be implicated in PCOS etiology and represents the major cause of cardiometabolic complications. The aim of present study was to investigate for the first time the association of lipin 1 gene polymorphisms with metabolic and hormonal profile in PCOS patients and controls. Into a case-control study 371 individuals were enrolled: 222 PCOS patients and 149 controls. Two lipin 1 gene polymorphisms were analyzed: rs11693809 (intron 1 SNP) and rs2716610 (intron 17 SNP) using fluorescent hydrolyzing probes. Body mass index, fasting plasma insulin and glucose along with androgen profile were measured in all subjects. Plasma lipids were measured in 93 patients and 43 controls and oral glucose test (OGTT) was performed on 68 PCOS patients. C/T heterozygotes for intron 1 SNP had significantly lower LDL-cholesterol than wild type C/C homozygotes (p=0.026) in the control group. In PCOS patients, mutated T/T homozygotes exhibited significantly lower glucose after OGTT than heterozygotes (p=0.033). Similarly, in nonobese PCOS patients, intron 1 SNP T/T homozygotes had lower HOMA-IR than heterozygotes (p=0.009). For intron 17 SNP, mutated C/T+T/T genotypes were associated with higher plasma triglycerides in controls (p=0.039). Genotype and allele frequencies were similar between PCOS patients and controls for both SNPs. Our results show that, in PCOS patients, intron 1 SNP is protective against IR and glucose intolerance suggesting that lipin 1 variation could be one of the genetic factors in cardiometabolic complications of PCOS.
Assuntos
Predisposição Genética para Doença , Fosfatidato Fosfatase/genética , Síndrome do Ovário Policístico/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Glicemia/metabolismo , Estudos de Casos e Controles , LDL-Colesterol/sangue , Feminino , Frequência do Gene/genética , Teste de Tolerância a Glucose , Haplótipos/genética , Humanos , Íntrons/genética , Fenótipo , Síndrome do Ovário Policístico/sangue , Triglicerídeos/sangue , Adulto JovemRESUMO
Impaired activity of the enzyme galactose-1-phosphate uridyltransferase (GALT) has been proposed as a risk factor for idiopathic presenile cataract. A study was undertaken to determine the prevalence of the three most common mutations in the GALT gene (Q188R, K285N and N314D, including its variant Duarte-2) in a group of Slovenian patients with idiopathic presenile cataract. GALT activity was determined in the erythrocytes of 30 cataract patients. DNA was isolated from their blood and analysed for Q188R, K285N and N314D mutations and IVS5-24G>A intronic variation by means of polymerase chain reaction and digestion with restriction enzymes. The average GALT activity of the cataract group was 19.5+/-4.9 U/g Hb, which is lower than the normal range (p = 0.034). Frequencies of Q188R, K285N, N314D and Duarte-2 alleles in the cataract group were 0.00%, 5.0%, 11.7% and 3.3%, respectively. Only the frequency of the K285N mutation was significantly higher in the patient group than in the control group (p = 0.0244). Our results support the reported association of decreased GALT activity with idiopathic presenile cataract. Molecular analysis indicates that, in the Slovenian population, this association is linked to the K285N mutation, which is neonatally benign in heterozygotes.
Assuntos
Catarata/enzimologia , Catarata/genética , Mutação/fisiologia , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Adulto , Alelos , DNA/genética , DNA/isolamento & purificação , Feminino , Galactitol/sangue , Galactosemias/genética , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
1. The modulation of synaptic transmission by serotonin (5-HT) was studied using whole-cell voltage-clamp and sharp-electrode current-clamp recordings from CA1 pyramidal neurones in transverse rat hippocampal slices in vitro. 2. With GABA(A) receptors blocked, polysynaptic transmission evoked by stratum radiatum stimulation was inhibited by submicromolar concentrations of 5-HT, while monosynaptic excitatory transmission and CA1 pyramidal neurone excitability were unaffected. The effect persisted following pharmacological blockade of 5-HT(1A) and 5-HT(4) receptors, which directly affect CA1 pyramidal neurone excitability. 3. Concentration-response relationships for 5-HT were determined in individual neurones; the EC(50) values for block of polysynaptic excitation and inhibition by 5-HT were approximately 230 and approximately 160 nM, respectively. The 5-HT receptor type responsible for the observed effect does not fall easily into the present classification of 5-HT receptors. 4. 5-HT inhibition of polysynaptic EPSCs persisted following complete block of GABAergic transmission and in CA1 minislices, ruling out indirect effects through interneurones and non-CA1 pyramidal neurones, respectively. 5. Monosynaptic EPSCs evoked by stimulation of CA1 afferent pathways appeared to be unaffected by 5-HT. Monosynaptic EPSCs evoked by stimulation of the alveus, which contains CA1 pyramidal neurone axons, were partially inhibited by 5-HT. 6. We conclude that 5-HT inhibited synaptic transmission by acting at local recurrent collaterals of CA1 pyramidal neurones. This may represent an important physiological action of 5-HT in the hippocampus, since it occurs over a lower concentration range than the 5-HT effects reported so far.
Assuntos
Hipocampo/fisiologia , Inibição Neural , Receptores de Serotonina/fisiologia , Serotonina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Condutividade Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that is inhibited by adrenocorticotropic hormone (ACTH) at picomolar concentrations. Inhibition of IAC may be a critical step in depolarization-dependent Ca2+ entry leading to cortisol secretion. In whole-cell patch clamp recordings from AZF cells, we have characterized properties of IAC and the signalling pathway by which ACTH inhibits this current. IAC was identified as a voltage-gated, outwardly rectifying, K(+)-selective current whose inhibition by ACTH required activation of a pertussis toxin-insensitive GTP binding protein. IAC was selectively inhibited by the cAMP analogue 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (8-pcpt-cAMP) with an IC50 of 160 microM. The adenylate cyclase activator forskolin (2.5 microM) also reduced IAC by 92 +/- 4.7%. Inhibition of IAC by ACTH, 8-pcpt-cAMP and forskolin was not prevented by the cAMP-dependent protein kinase inhibitors H-89 (5 microM), cAMP-dependent protein kinase inhibitor peptide (PKI[5-24]) (2 microM), (Rp)-cAMPS (500 microM), or by the nonspecific protein kinase inhibitor staurosporine (100 nM) applied externally or intracellularly through the patch pipette. At the same concentrations, these kinase inhibitors abolished 8-pcpt-cAMP-stimulated A-kinase activity in AZF cell extracts. In intact AZF cells, 8-pcpt-cAMP activated A-kinase with an EC50 of 77 nM, a concentration 2,000-fold lower than that inhibiting IAC half maximally. The active catalytic subunit of A-kinase applied intracellularly through the recording pipette failed to alter functional expression of IAC. The inhibition of IAC by ACTH and 8-pcpt-cAMP was eliminated by substituting the nonhydrolyzable ATP analogue AMP-PNP for ATP in the pipette solution. Penfluridol, an antagonist of T-type Ca2+ channels inhibited 8-pcpt-cAMP-induced cortisol secretion with an IC50 of 0.33 microM, a concentration that effectively blocks Ca2+ channel in these cells. These results demonstrate that IAC is a K(+)-selective current whose gating is controlled by an unusual combination of metabolic factors and membrane voltage. IAC may be the first example of an ionic current that is inhibited by cAMP through an A-kinase-independent mechanism. The A-kinase-independent inhibition of IAC by ACTH and cAMP through a mechanism requiring ATP hydrolysis appears to be a unique form of channel modulation. These findings suggest a model for cortisol secretion wherein cAMP combines with two separate effectors to activate parallel steroidogenic signalling pathways. These include the traditional A-kinase-dependent signalling cascade and a novel pathway wherein cAMP binding to IAC K+ channels leads to membrane depolarization and Ca2+ entry. The simultaneous activation of A-kinase- and Ca(2+)-dependent pathways produces the full steroidogenic response.
Assuntos
Trifosfato de Adenosina/metabolismo , Córtex Suprarrenal/fisiologia , Hormônio Adrenocorticotrópico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/farmacologia , Potássio/fisiologia , Córtex Suprarrenal/citologia , Animais , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Condutividade Elétrica , Proteínas de Ligação ao GTP/fisiologia , Hidrocortisona/metabolismo , Hidrólise , Cinética , Masculino , Nucleotídeos Cíclicos/farmacologia , Potássio/antagonistas & inibidores , Proteínas Quinases/fisiologiaRESUMO
In bovine adrenal zona fasciculata (AZF) cells, angiotensin II (AII) may stimulate depolarization-dependent Ca2+ entry and cortisol secretion through inhibition of a novel potassium channel (IAC), which appears to set the resting potential of these cells. Aspects of the signaling pathway, which couples AII receptors to membrane depolarization and secretion, were characterized in patch clamp and membrane potential recordings and in secretion studies. AII-mediated inhibition of IAC, membrane depolarization, and cortisol secretion were all blocked by the AII type I (AT1) receptor antagonist losartan. These responses were unaffected by the AT2 antagonist PD123319. Inhibition of IAC by AII was prevented by intracellular application of guanosine 5'-O-2-(thio)-diphosphate but was not affected by pre-incubation of cells with pertussis toxin. Although mediated through an AT1 receptor, several lines of evidence indicated that AII inhibition of IAC occurred through an unusual phospholipase C (PLC)-independent pathway. Acetylcholine, which activates PLC in AZF cells, did not inhibit IAC. Neither the PLC antagonist neomycin nor PLC-generated second messengers prevented IAC expression or mimicked the inhibition of this current by AII. IAC expression and inhibition by AII were insensitive to variations in intracellular or extracellular Ca2+ concentration. AII-mediated inhibition of IAC was markedly reduced by the non-hydrolyzable ATP analog adenosine 5'-(beta, gamma-imino)triphosphate and by the non-selective protein kinase inhibitor staurosporine. The protein phosphatase antagonist okadaic acid reversibly inhibited IAC in whole cell recordings. These findings indicate that AII-stimulated effects on IAC current, membrane voltage, and cortisol secretion are linked through a common AT1 receptor. Inhibition of IAC in AZF cells appears to occur through a novel signaling pathway, which may include a losartan-sensitive AT1 receptor coupled through a pertussis-insensitive G protein to a staurosporine-sensitive protein kinase. Apparently, the mechanism linking AT1 receptors to IAC inhibition and Ca2+ influx in adrenocortical cells is separate from that involving inositol trisphosphate-stimulated Ca2+ release from intracellular stores. AII-stimulated cortisol secretion may occur through distinct parallel signaling pathways.
Assuntos
Glândulas Suprarrenais/metabolismo , Compostos de Bifenilo/farmacologia , Hidrocortisona/metabolismo , Imidazóis/farmacologia , Receptores de Angiotensina/metabolismo , Transdução de Sinais , Tetrazóis/farmacologia , Glândulas Suprarrenais/fisiologia , Angiotensina II/farmacologia , Animais , Bovinos , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Nucleotídeos de Guanina/metabolismo , Técnicas In Vitro , Ativação do Canal Iônico , Losartan , Potenciais da Membrana , Fosfolipases/metabolismo , Fosforilação , Bloqueadores dos Canais de Potássio , Proteínas Quinases/metabolismo , Receptores de Angiotensina/efeitos dos fármacosRESUMO
We have studied the interaction of dihydropyridine (DHP) Ca2+ channel agonists and antagonists with A-type K+ channels in whole-cell patch-clamp recordings from bovine adrenal zona fasciculata cells. At concentrations from 1 to 100 microM, DHP antagonists [nimodipine and (+)-Bay K 8644] and agonists [(-)-Bay K 8644 and RS 30026] each reversibly reduced A-type K+ current (IA) amplitude and markedly accelerated the apparent rate of IA inactivation. Unlike their actions on Ca2+ channels, the effects of DHP agonists and antagonists on IA were qualitatively indistinguishable. Inhibition of IA by DHPs was not accompanied by changes in the voltage-dependent steady state inactivation of IA or the kinetics of recovery subsequent to repolarization. The effects of DHPs on peak IA and inactivation kinetics were not use dependent. The DHPs were much less effective in cells where fast N-type inactivation had spontaneously diminished with time. These actions of DHPs on IA are in marked contrast to their voltage-dependent modulation of L-type Ca2+ currents, indicating that fundamentally different mechanisms are involved. Rather than directly occluding A-type K+ channels, the drugs may enhance the voltage-independent rate of inactivation. This could occur through interaction of the DHP with a site on the amino-terminal inactivation domain or the DHP binding site at the inner mouth of the channel. Regardless of the mechanism involved, the identical modulation by DHP agonists and antagonists is a distinctive feature of A-type K+ channels in adrenal zona fasciculata cells.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Bloqueadores dos Canais de Potássio , Animais , Bovinos , Células Cultivadas , Potenciais da Membrana/efeitos dos fármacos , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/metabolismo , Zona Fasciculada/fisiologiaRESUMO
1. The effects of the trivalent cations yttrium (Y3+), lanthanum (La3+), cerium (Ce3+), neodymium (Nd3+), gadolinium (Gd3+), holmium (Ho3+), erbium (Er3+), ytterbium (Yb3+) and the divalent cation nickel (Ni2+) on the T-type voltage gated calcium channel (VGCC) were characterized by the whole-cell patch clamp technique using rat and human thyroid C cell lines. 2. All the metal cations (M3+) studied, blocked current through T-type VGCC (IT) in a concentration-dependent manner. Smaller trivalents were the best T-channel antagonists and potency varied inversely with ionic radii for the larger M3+ ions. Estimation of half-maximal blocking concentrations (IC50s) for IT carried by 10 mM Ca2+ resulted in the following potency sequence: Ho3+ (IC50 = 0.107 microM) approximately Y3+ (0.117) approximately Yb3+ (0.124) > or = Er3+ (0.153) > Gd3+ (0.267) > Nd3+ (0.429) > Ce3+ (0.728) > La3+ (1.015) >> Ni2+ (5.65). 3. Tail current measurements and conditioning protocols were used to study the influence of membrane voltage on the potency of these antagonists. Block of IT by Ni2+, Y3+, La3+ and the lanthanides was voltage independent in the range from -200 to +80 mV. In addition, the antagonists did not affect macroscopic inactivation and deactivation of T-type VGCC. 4. Increasing the extracellular Ca2+ concentration reduced the potency of IT block by Ho3+, indicative of competitive antagonism between this blocker and the permeant ion for a binding site. 5. The results suggest that the mechanism of metal cation block of T-type VGCC is occlusion of the channel pore by the antagonist binding to a Ca2+/M3+ binding site, located out of the membrane electric field. 6. Block of T-type VGCC by Y3+, lanthanides and La3+ differ from the inhibition of high voltage-activated VGCC block in several respects: smaller cations are more potent IT antagonists; block is voltage independent and the antagonists do not permeate T-type channels. These differences suggest corresponding structural dissimilarities in the permeation pathways of low and high voltage-activated Ca2+ channels.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cátions/farmacologia , Neurônios/metabolismo , Níquel/farmacologia , Glândula Tireoide/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Eletrofisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Lantânio/farmacologia , Metais Terras Raras/farmacologia , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Células Tumorais Cultivadas , Itérbio/farmacologiaRESUMO
The whole cell version of the patch clamp technique was used to identify and characterize voltage-gated Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells. The great majority of cells (84 of 86) expressed only low voltage-activated, rapidly inactivating Ca2+ current with properties of T-type Ca2+ current described in other cells. Voltage-dependent activation of this current was fit by a Boltzmann function raised to an integer power of 4 with a midpoint at -17 mV. Independent estimates of the single channel gating charge obtained from the activation curve and using the "limiting logarithmic potential sensitivity" were 8.1 and 6.8 elementary charges, respectively. Inactivation was a steep function of voltage with a v1/2 of -49.9 mV and a slope factor K of 3.73 mV. The expression of a single Ca2+ channel subtype by AZF cells allowed the voltage-dependent gating and kinetic properties of T current to be studied over a wide range of potentials. Analysis of the gating kinetics of this Ca2+ current indicate that T channel activation, inactivation, deactivation (closing), and reactivation (recovery from inactivation) each include voltage-independent transitions that become rate limiting at extreme voltages. Ca2+ current activated with voltage-dependent sigmoidal kinetics that were described by an m4 model. The activation time constant varied exponentially at test potentials between -30 and +10 mV, approaching a voltage-independent minimum of 1.6 ms. The inactivation time constant (tau i) also decreased exponentially to a minimum of 18.3 ms at potentials positive to 0 mV. T channel closing (deactivation) was faster at more negative voltages; the deactivation time constant (tau d) decreased from 8.14 +/- 0.7 to 0.48 +/- 0.1 ms at potentials between -40 and -150 mV. T channels inactivated by depolarization returned to the closed state along pathways that included two voltage-dependent time constants. tau rec-s ranged from 8.11 to 4.80 s when the recovery potential was varied from -50 to -90 mV, while tau rec-f decreased from 1.01 to 0.372 s. At potentials negative to -70 mV, both time constants approached minimum values. The low voltage-activated Ca2+ current in AZF cells was blocked by the T channel selective antagonist Ni2+ with an IC50 of 20 microM. At similar concentrations, Ni2+ also blocked cortisol secretion stimulated by adrenocorticotropic hormone. Our results indicate that bovine AZF cells are distinctive among secretory cells in expressing primarily or exclusively T-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Canais de Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Zona Fasciculada/citologia , Zona Fasciculada/fisiologia , Animais , Bovinos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Células Cultivadas , Potenciais da Membrana/fisiologia , Níquel/farmacologia , Zona Fasciculada/ultraestruturaRESUMO
In whole cell patch clamp recordings on enzymatically dissociated adrenal zona fasciculata (AZF) cells, a rapidly inactivating A-type K+ current was observed in each of more than 150 cells. Activation of IA was steeply voltage dependent and could be described by a Boltzmann function raised to an integer power of 4, with a midpoint of -28.3 mV. Using the "limiting logarithmic potential sensitivity," the single channel gating charge was estimated to be 7.2 e. Voltage-dependent inactivation could also be described by a Boltzmann function with a midpoint of -58.7 mV and a slope factor of 5.92 mV. Gating kinetics of IA included both voltage-dependent and -independent transitions in pathways between closed, open, and inactivated states. IA activated with voltage-dependent sigmoidal kinetics that could be fit with an n4h formalism. The activation time constant, tau a, reached a voltage-independent minimum at potentials positive to 0 mV. IA currents inactivated with two time constants that were voltage independent at potentials ranging from -30 to +45 mV. At +20 mV, tau i(fast) and tau i(slow) were 13.16 +/- 0.64 and 62.26 +/- 5.35 ms (n = 34), respectively. In some cells, IA inactivation kinetics slowed dramatically after many minutes of whole cell recording. Once activated by depolarization, IA channels returned to the closed state along pathways with two voltage-dependent time constants which were 0.208 s, tau rec-f and 10.02 s, tau rec-s at -80 mV. Approximately 90% of IA current recovered with slow kinetics at potentials between -60 and -100 mV. IA was blocked by 4-aminopyridine (IC50 = 629 microM) through a mechanism that was strongly promoted by channel activation. Divalent and trivalent cations including Ni2+ and La3+ also blocked IA with IC50's of 467 and 26.4 microM, respectively. With respect to biophysical properties and pharmacology, IA in AZF cells resembles to some extent transient K+ currents in neurons and muscle, where they function to regulate action potential frequency and duration. The function of this prominent current in steroid hormone secretion by endocrine cells that may not generate action potentials is not yet clear.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio/fisiologia , Zona Fasciculada/citologia , Zona Fasciculada/fisiologia , Aminopiridinas/farmacologia , Animais , Bovinos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Potenciais da Membrana/fisiologia , Níquel/farmacologia , Zona Fasciculada/ultraestruturaRESUMO
The function of low voltage-activated T-type Ca2+ channels in ACTH-stimulated cortisol production by bovine adrenal zona fasciculata cells (AZF) was explored in patch clamp and secretion studies. Nearly all AZF cells expressed only a low voltage-activated T-type Ca2+ current (IT) that was blocked by the diphenylbutylpiperidine (DPBP) Ca2+ antagonists penfluridol and pimozide with IC50S of 0.3 and 0.5 microM, respectively. Dihydropyridine (DHP) Ca2+ antagonists, including nimodipine, nisoldipine, and felodipine, also blocked T-type Ca2+ current with IC50S ranging from 3.5-8.8 microM. Inhibition of IT by DPBP and DHP antagonists was voltage and use dependent. ACTH (1 nM) stimulated large (> 50-fold) increases in cortisol production by AZF cells, which were inhibited by Ca2+ antagonists at concentrations similar to those which blocked IT. Inhibition of cortisol production by Ca2+ antagonists was specific; ACTH-induced insulin-like growth factor-I production by AZF cells was not affected by DPBP antagonists. The L channel-specific DHP Ca2+ agonist (-)Bay K 8644 did not enhance basal or ACTH-stimulated cortisol synthesis. These results demonstrate that functional T-rather than L-type Ca2+ channels are required for ACTH-stimulated cortisol synthesis. They also suggest that these low voltage-activated channels, acting as the primary pathway for Ca2+ entry into AZF cells, couple ACTH-stimulated membrane depolarization to steroid hormone production.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Canais de Cálcio/fisiologia , Cálcio/fisiologia , Hidrocortisona/biossíntese , Zona Fasciculada/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/classificação , Bovinos , Células Cultivadas , AMP Cíclico/fisiologia , Di-Hidropiridinas/farmacologia , Hidrocortisona/metabolismo , Fator de Crescimento Insulin-Like I/biossíntese , Masculino , Penfluridol/farmacologia , Pimozida/farmacologia , Transdução de Sinais , Zona Fasciculada/metabolismoRESUMO
Adrenocorticotropic hormone (ACTH) and angiotensin II (AII) are peptides that regulate the production of steroid hormones by cells of the adrenal cortex. The cellular mechanisms linking these peptides to corticosteroid hormone secretion are not understood. In patch clamp recordings from bovine adrenal zona fasciculata (AZF) cells, we have identified a novel cholera toxin-sensitive K+ current (IAC), which is potently inhibited by both ACTH and AII with respective EC50 values of 4.5 and 145 pM. These two peptides depolarize AZF cells with a temporal pattern and potency that parallels the inhibition of IAC. With the discovery of IAC, we have identified a common molecular target for both ACTH and AII. The convergent inhibition of IAC by these two peptides suggests a mechanism whereby biochemical signals originating at the cell membrane can be transduced to depolarization-dependent Ca2+ entry and steroid hormone secretion.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Canais de Potássio/metabolismo , Córtex Suprarrenal/citologia , Corticosteroides/metabolismo , Animais , Bovinos , Células Cultivadas , Cinética , Potenciais da Membrana , Potássio/metabolismoRESUMO
The whole cell version of the patch-clamp technique was used to identify and characterize voltage-gated Ca2+, Na+, and K+ currents in the calcitonin-secreting human thyroid TT cell line. Ca2+ current consisted of a single low-voltage-activated rapidly inactivating component. The current was one-half maximally activated at a potential of -27 mV, while steady-state voltage-dependent inactivation was one-half complete at -51 mV. The Ca2+ current inactivated with a voltage-dependent time constant that reached a minimum of 16 ms at potentials positive to -15 mV. Deactivation kinetics could also be fit with a single voltage-dependent time constant of approximately 2 ms at -80 mV. Replacing Ca2+ with Ba2+ reduced the maximum current by 18 +/- 5% (n = 6). The dihydropyridine Ca2+ agonist (-)BAY K 8644 did not affect the Ca2+ current, but 50 microM Ni2+ reduced it by 81 +/- 0.8% (n = 5). TT cells also possessed tetrodotoxin-sensitive voltage-gated Na+ channels and tetraethylammonium-sensitive delayed rectifier type K+ currents. These results indicate that TT cells possess membrane currents necessary for the generation of action potentials. T-type Ca2+ channels are the sole pathway for voltage-dependent Ca2+ entry into these cells and may couple electrical activity to calcitonin secretion.
Assuntos
Calcitonina/metabolismo , Carcinoma/fisiopatologia , Neoplasias da Glândula Tireoide/fisiopatologia , Carcinoma/metabolismo , Carcinoma/patologia , Eletrofisiologia , Humanos , Cinética , Potássio/fisiologia , Sódio/fisiologia , Tetrodotoxina/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
We have used the whole-cell version of the patch-clamp technique to analyze the inhibition of Ca2+ currents by antipsychotic agents in neural crest-derived rat and human thyroid C cell lines. Diphenylbutylpiperidine (DPBP) antipsychotics, including penfluridol and fluspirilene, potently and preferentially block T-type Ca2+ current in the rat medullary thyroid carcinoma 6-23 (clone 6) cell line. When step depolarizations were applied at 0.1 Hz from a holding potential of -80 mV, with 10 mM Ca2+ as the charge carrier, the DPBP penfluridol inhibited T-type current with an IC50 of 224 nM. High voltage-activated L and N currents were less potently blocked. At a concentration of 500 nM, penfluridol inhibited 78.0 +/- 2.3% (n = 29) of inactivating T-type Ca2+ current, whereas the sustained high voltage-activated current was reduced by 25.6 +/- 3.5% (n = 28). Block of T-type current by penfluridol was enhanced by depolarizing test pulses applied at frequencies above 0.03 Hz. The use-dependent component of block was largely reversed by pulse-free periods at -80 mV. T-type Ca2+ channels in the human TT C cell line were blocked by penfluridol, and the potency was enhanced by reduction of extracellular Ca2+. Non-DPBP antipsychotics, including haloperidol, clozapine, and thioridazine, also blocked T-type channels, but these were 20-100 times less potent than the DPBPs. These results identify the DPBPs as a new class of organic Ca2+ channel antagonists, which are distinctive in their ability to preferentially block T-type channels. These agents will be useful in defining the function of T channels in various excitable cells. Their potent block of T-type Ca2+ channels, which would be enhanced in rapidly firing cells, suggests that this action may be relevant to the therapeutic or toxic effects of these drugs when used in clinical pharmacology.
Assuntos
Antipsicóticos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Crista Neural/efeitos dos fármacos , Animais , Canais de Cálcio/fisiologia , Eletrofisiologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Crista Neural/patologia , Crista Neural/fisiologia , Penfluridol/farmacologia , Ratos , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/fisiopatologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
We have studied the interaction between dihydropyridine (DHP) Ca2+ modulators and the phorbol ester phorbol 12-myristate 13-acetate (PMA) on whole cell Ca2+ currents, 45Ca2+ uptake, immediate early gene (IEG) expression, and proliferation in the rat pituitary GH4C1 cell line. When short (3- to 5-msec) depolarizing voltage clamp steps were used to activate L-type Ca2+ channels, the DHP Ca2+ agonist (-)Bay K 8644 markedly enhanced Ca2+ entry by slowing channel closing upon repolarization. In contrast, the Ca2+ agonist induced only small and inconsistent increases in c-fos mRNA and did not measurably increase NGFI-A. Ca2+ channel activation by depolarization with 50 mM KCl in the presence of (-)Bay K 8644 induced large increases in 45Ca2+ uptake, but failed to markedly induce either of the IEGs. The phorbol ester PMA did not alter T- or L-type Ca2+ current or 45Ca2+ uptake by GH4C1 cells, but triggered large increases in both c-fos and NGFI-A mRNA. In combination, PMA and (-)Bay K 8644 acted synergistically to increase mRNAs for both IEGs. The effect of the DHPs was stereospecific; (+)Bay K 8644, a Ca2+ antagonist, inhibited PMA-induced increases in c-fos and NGFI-A mRNAs. Both PMA and (-)Bay K 8644 inhibited the proliferation of GH4C1 cells, measured by cell count or [3H]thymidine incorporation. The inhibition by the Ca2+ agonist was stereoselective and approximately additive to that of PMA. These results indicate that the expression of c-fos IEG and that of NGFI-A IEG are differentially regulated by separate second messenger pathways in GH4C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)