RESUMO
Testosterone (TES), other androgens and female sex steroids induce non-genomic rapid relaxing effects in airway smooth muscle (ASM). In guinea pig ASM, basal tension was relaxed by dehydroepiandrosterone (DHEA) and TES; 17ß-estradiol (E2) had a small effect. Blockers of L-type voltage dependent Ca2+ channel (L-VDCC, D-600) and store operated Ca2+ channel (SOC, 2-APB) also relaxed the basal tone. In tracheal myocytes, DHEA and TES diminished intracellular basal Ca2+ concentrations (b[Ca2+]i) as D-600+2-APB but to a higher extend. TES after D-600+2APB or Pyr3, a blocker of canonical transient receptor potential 3 (TRPC3), further decreased b[Ca2+]i rendering this response equal to TES alone. With indomethacin, the b[Ca2+]i decrease induced by the blockade of L-VDCC and TRPC3 was not changed by the addition of TES. PGE2 or forskolin addition after D600+2-APB, decreased b[Ca2+]i resembling TES response. An adenylate cyclase inhibitor followed by D-600+2-APB lowered b[Ca2+]i, TES showed no further effect. Carbachol-induced [Ca2+]i increment was reduced by TES or DHEA. 17ß-estradiol diminished KCl-induced contraction and, in tracheal myocytes, the voltage-dependent inward Ca2+ current. CONCLUSION: DHEA and TES diminish ASM tone and b[Ca2+]i by blocking L-VDCC and probably a constitutively active TRPC3, and by PGE2 synthesis. E2 lowers ASM basal tone by blocking only L-VDCC.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Hormônios Esteroides Gonadais/farmacologia , Espaço Intracelular/metabolismo , Músculo Liso/fisiologia , Traqueia/fisiologia , Animais , Compostos de Boro/farmacologia , Carbacol/farmacologia , AMP Cíclico/metabolismo , Desidroepiandrosterona/farmacologia , Estradiol/farmacologia , Galopamil/farmacologia , Cobaias , Masculino , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso/efeitos dos fármacos , Prostaglandinas/metabolismo , Canais de Cátion TRPC/metabolismo , Testosterona/farmacologiaRESUMO
An increase in intracellular calcium is essential to trigger capacitation and the acrosome reaction. The aim of this study was to determine the progesterone effect mediated by the voltage-dependent calcium channel and protein kinase C on heparin-capacitated and noncapacitated spermatozoa. Protein kinase C was activated by 1-oleoyl-2-acetyl glycerol, a membrane-permeant diacyl-glycerol, and inhibited by GF-109203X. The percentage of true acrosome reaction was evaluated using differential-interferential optical contrast microscopy and trypan blue stain. The calcium concentration was evaluated by FURA-2AM and methoxyverapamil was used as a voltage-dependent calcium channel inhibitor. A rapid calcium increase and acrosome reaction were induced by progesterone in capacitated and noncapacitated spermatozoa, a higher intracellular calcium increase being observed in capacitated than in noncapacitated samples (P < 0.05). The calcium increase and acrosome reaction were blocked significantly by GF-109203X in noncapacitated and capacitated spermatozoa by the addition of progesterone and/or 1-oleoyl-2-acetylglycerol. Methoxyverapamil blocked calcium influx in samples treated with progesterone and heparin/progesterone, but not in those treated with 1-oleoyl-2-acetyl glycerol. Progesterone induces the acrosome reaction in noncapacitated cryopreserved bovine spermatozoa through intracellular mechanisms dependent on protein kinase C and the voltage-dependent calcium channel.
Assuntos
Canais de Cálcio/fisiologia , Criopreservação , Progesterona/farmacologia , Proteína Quinase C/metabolismo , Espermatozoides/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bovinos , Diglicerídeos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Galopamil/farmacologia , Heparina/farmacologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Preservação do Sêmen , Capacitação Espermática , Espermatozoides/fisiologiaRESUMO
BACKGROUND: In airway smooth muscle (ASM), Ca2+ influx in response to the Ca2+ depletion of the sarcoplasmic reticulum (SR) seems to play a role in the regulation of intracellular free Ca2+ concentrations ([Ca2+](i)). This study evaluates some possible Ca2+ entry pathways activated during SR-Ca2+ depletion induced by 10 mM caffeine. METHODS: Enzymatically dispersed bovine ASM cells were loaded with Fura-2/AM to permit measurement of [Ca2+](i) changes in single cells. RESULTS: Caffeine (10 mM) induced a transient increase in [[Ca2+](i) that depleted SR-Ca(2)+ content. After caffeine washout, a decrease in basal [Ca2+](i) (undershoot) was invariably observed, followed by a slow recovery. This phenomenon was inhibited by cyclopiazonic acid (5 microM). External Ca(2)+ removal in depolarized and nondepolarized cells induced a decrease in basal [Ca2+](i) that continued until depletion of the SR-Ca2+ content. The decrease in [Ca2+](i) induced by Ca2+-free physiological saline solution (PSS) was accelerated in caffeine-stimulated cells. Recovery from undershoot was not observed in Ca2+-free PSS. Depolarization with KCl and addition of D600 (30 microM) did not modify recovery. Similar results were obtained when the Na(+)/Ca2+ exchanger was blocked by substituting NaCl with KCl in normal PSS (Na(+)-free PSS) or by adding benzamil amiloride (25 microM). CONCLUSIONS: SR-Ca2+ content plays an important role in the Ca2+ leak induced by Ca2+-free medium, and does not depend on membrane potential. Additionally, recovery from undershoot after caffeine depends on extracellular Ca2+, and neither voltage-dependent Ca2+ channels nor the Na(+)/Ca2+ exchanger are involved.
Assuntos
Cafeína/farmacologia , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Músculo Liso/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Traqueia/citologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Canais de Cálcio/fisiologia , Carbacol/farmacologia , Bovinos , Células Cultivadas , Galopamil/farmacologia , Indóis/farmacologia , Transporte de Íons/efeitos dos fármacos , Ionomicina/farmacologia , Ionóforos/farmacologia , Músculo Liso/citologia , Cloreto de Potássio/farmacologia , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/efeitos dos fármacos , Trocador de Sódio e Cálcio/fisiologiaRESUMO
Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na+, K+, and Ca2+ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca2+ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca2+ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca2+, Ba2+, Sr2+, Mn2+, and Co2+. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La3+ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca2+ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca2+ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca2+ pools required for virus maturation and cell death.
Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular , Rotavirus/fisiologia , Animais , Células Cultivadas , Galopamil/farmacologia , Haplorrinos , Lantânio/farmacologia , Metais/metabolismoRESUMO
Rotavirus infection modifies the metabolism and ionic homeostasis of the host cell. First, there is an induction of viral synthesis with a parallel shutoff of cell protein production, followed by an increase of plasma membrane Ca2+ permeability, thereby inducing an increase of free cytoplasmic and sequestered Ca2+ concentrations. Cell death follows at a later stage. We studied the role of the increase in Ca2+ concentration in cell death. An elevation of extracellular Ca2+ concentration during infection induced an increase in [Ca2+]i and potentiated cell death. Buffering the increases in [Ca2+]i with BAPTA added at 6 h p.i. reduced the cytopathic effect without inhibiting viral protein synthesis and infectious particle production. Metoxyverapamil (D600), a Ca2+ channel inhibitor, added at 1 h p.i. reduced Ca2+ permeability, the increases in [Ca2+]i, and cell death produced by infection without modifying viral protein synthesis and infectious titer. Thapsigargin, the inhibitor of Ca(2+)-ATPase of endoplasmic reticulum, potentiated the increase of [Ca2+]i and accelerated the time course of cell death. Double staining with fluorescein diacetate and ethidium bromide or acridine orange and ethidium bromide showed that infected MA104 cells had lost plasma membrane integrity without DNA fragmentation or formation of apoptotic bodies. These results support the hypothesis that the increase in [Ca2+]i due to a product of viral protein synthesis triggers the chain of events that leads to cell death by oncosis.
Assuntos
Cálcio/metabolismo , Morte Celular , Infecções por Rotavirus/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Quelantes/farmacologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Galopamil/farmacologia , Haplorrinos , Homeostase , Microscopia de Fluorescência , Infecções por Rotavirus/patologia , Tapsigargina/farmacologia , Proteínas Virais/biossínteseRESUMO
The effect of nitric oxide donors on intracellular calcium concentration [Ca2+]i was studied in anterior pituitary cells using ratiometric FURA 2 fluorescence measurements. Sodium nitroprusside (NP) induced a transient decrease in [Ca2+]i, after which [Ca2+]i returned to, or even increased over basal values. S-Nitroso glutathione (GSNO) induced a similar decrease. NP also inhibited high [Ca2+]i achieved by depolarization with 25 mM K+. The inhibitory effect of NP was partially blunted by pretreatment with methoxy-verapamil, and in calcium free buffer, and was not altered by thapsigargin. Interestingly, in calcium free buffer there was a significant stimulatory effect of NP, which was partially blunted by thapsigargin. We conclude that NO donors modify [Ca2+]i in anterior pituitary cells. The action is biphasic, with an initial decrease in [Ca2+]i probably related to a decrease of Ca2+ influx through VDCC, and an increase evidenced in calcium free buffer in which the inhibitory component is absent, and partially depends on thapsigargin sensitive calcium stores.
Assuntos
Cálcio/metabolismo , Doadores de Óxido Nítrico/farmacologia , Adeno-Hipófise/metabolismo , Animais , Soluções Tampão , Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/farmacologia , Galopamil/farmacologia , Glutationa/análogos & derivados , Glutationa/farmacologia , Líquido Intracelular/metabolismo , Cinética , Masculino , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , Potássio/farmacologia , Ratos , Ratos Wistar , S-Nitrosoglutationa , Tapsigargina/farmacologiaRESUMO
This report investigates the mechanisms by which heparin induces capacitation of sperm. Capacitation was determined with chlortetracycline and intracellular calcium concentration, [Ca2+]i, with FURA 2-AM. After 15 minutes incubation with heparin [Ca2+]i was increased 60% over basal, reaching a plateau thereafter. Sixty percent of calcium entry was inhibited by methoxy-verapamil, suggesting that activation of voltage dependent calcium channels (VDCC) may be involved in the process. A significant correlation (R1 = 0.88, p < 0.006) was found between the percentage of capacitated spermatozoa and the [Ca2+]i increase. The effects of heparin on both processes were blocked by the protein kinase C (PKC) inhibitors staurosporine (100%) and GF-109203X (90%). It is concluded that heparin may induce sperm capacitation and calcium influx mainly through VDCC (approx. 60%), and also through other membrane systems (40%). Both systems of calcium entry as well as the capacitation process appear to involve on PKC activity.
Assuntos
Cálcio/metabolismo , Heparina/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Transporte Biológico , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Bovinos , Inibidores Enzimáticos/farmacologia , Galopamil/farmacologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Transdução de Sinais , Estaurosporina/farmacologiaRESUMO
Canatoxin (CNTX), a toxic protein isolated from seeds of Canavalia ensiformis, induces Ca2+ influx across the platelet plasma membrane, mobilization of arachidonic acid mediated by phospholipase A2, ATP secretion, and platelet aggregation. All these events depend on the presence of extracellular Ca2+ and are blocked by methoxyverapamil, a calcium-channel blocker. CNTX does not activate phospholipase C, and the intracellular calcium mobilization mediated by IP3 does not play a role in platelet activation by this toxin. Preincubation of rabbit platelets with 8-Br-cGMP inhibited the CNTX-evoked calcium influx, arachidonate release, ATP secretion, and cell aggregation. Our data suggest that the calcium influx is a prior step on platelet activation by CNTX, being modulated by cGMP.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , GMP Cíclico/metabolismo , Galopamil/antagonistas & inibidores , Lectinas/farmacologia , Proteínas de Plantas , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Toxinas Biológicas , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Membrana Celular/metabolismo , Regulação para Baixo , Interações Medicamentosas , Galopamil/farmacologia , Transporte de Íons/efeitos dos fármacos , CoelhosRESUMO
The possible modifications of extracellular pH associated with the secretion of catecholamines evoked by the introduction of 2.2 mM Sr2+ to a Ca(2+)-free, buffer-free, Locke solution were investigated in decorticated perfused bovine adrenal glands. A progressive and reversible decrease of external pH accompanied the catecholamine release promoted by Sr(2+)-introduction into the perfusion fluid. This extracellular acid shift was practically undetected when the chromaffin tissue was stimulated by the addition of Sr2+ to a buffered medium. Both the secretory response as well as the extracellular pH drop mediated by Sr(2+)-introduction to a Ca(2+)-free, buffer-free, Locke solution were markedly inhibited by methoxyverapamil (0.3 mM), Mg2+ (20 mM) and hyperosmolarity (750 mOsm). The exposure of the adrenal medulla to a Ca(2+)-free, buffer-free, high-K+ solution containing 2.2 mM Sr2+ for 6 min promoted a significant enhancement of both the secretory response and the acidification of the perfusates compared with the responses evoked by Sr2+ in a 5.6 mM K+ medium. These results are consistent with the existence of a close relationship between extracellular acidification and the release of catecholamines triggered by the introduction of Sr2+ to the perfusion fluid.
Assuntos
Medula Suprarrenal/efeitos dos fármacos , Catecolaminas/metabolismo , Espaço Extracelular/metabolismo , Estrôncio/farmacologia , Medula Suprarrenal/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Bovinos , Galopamil/farmacologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Magnésio/metabolismo , Concentração Osmolar , Potássio/metabolismoRESUMO
The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.