RESUMO
We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.
Assuntos
Ácido Araquidônico/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Esôfago/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Músculo Liso/fisiologia , Receptores Muscarínicos/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Acetilcolina/farmacologia , Animais , Gatos , Diaminas/farmacologia , Feminino , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Parassimpatolíticos/farmacologia , Receptor Muscarínico M2RESUMO
1. This study evaluated the inhibitory action of apigenin-7-O-beta-D-glucuronopyranoside (AGC), apigenin, and omeprazole on reflux oesophagitis and gastritis in rats. AGC was isolated from Clerodendron trichotomum leaves. 2. Oesophagitis and gastritis were induced by surgical procedure and the administration of indomethacin, respectively. The intraduodenal (i.d.) administration of AGC decreased the volume of gastric juice and increased the gastric pH compared with apigenin and omeprazole. The acid output was more inhibited by AGC in a dose-dependent manner than by apigenin and omeprazole. Compared with apigenin and omeprazole, AGC significantly decreased the size of gastric lesions, which were induced by exposure of the gastric mucosa to indomethacin. 3. Malondialdehyde (MDA) content, which is the end product of lipid peroxidation, was increased significantly after the induction of reflux oesophagitis. The MDA content was decreased by AGC (i.d. 3 mg kg(-1)), but not by either apigenin or omeprazole. This suggests that AGC has an antioxidative effect. In the oesophagitis group, the mucosal levels of glutathione (GSH) were significantly lower than that in the normal group. However, the GSH levels were preserved after administering the AGC, suggesting that AGC possesses scavenging activity. 4. In summary, AGC is more potent than apigenin and omeprazole at inhibiting reflux oesophagitis and gastritis and may therefore be a promising drug for their treatment.
Assuntos
Apigenina/uso terapêutico , Esofagite Péptica/tratamento farmacológico , Gastrite/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/toxicidade , Antiulcerosos/uso terapêutico , Clerodendrum/química , Relação Dose-Resposta a Droga , Esofagite Péptica/etiologia , Esôfago/efeitos dos fármacos , Esôfago/metabolismo , Esôfago/patologia , Gastrite/induzido quimicamente , Glutationa/metabolismo , Indometacina/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Omeprazol/uso terapêutico , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Ratos , Ratos Sprague-DawleyRESUMO
1 We examined whether extremely low frequency electromagnetic fields (ELF-EMF) affect the basal level of cardiovascular parameters and influence of drugs acting on the sympathetic nervous system. 2 Male rats were exposed to sham control and EMF (60 Hz, 20 G) for 1 (MF-1) or 5 days (MF-5). We evaluated the alterations of blood pressure (BP), pulse pressure (PP), heart rate (HR), and the PR interval, QRS interval and QT interval on the electrocardiogram and dysrhythmic ratio in basal level and dysrhythmia induced by beta-adrenoceptor agonists. 3 In terms of the basal levels, there were no statistically significant differences among control, MF-1 and MF-5 in PR interval, QRS interval, mean BP, HR and PP. However, the QT interval, representing ventricular repolarization, was significantly reduced by MF-1 (P < 0.05). 4 (-)-Dobutamine (beta1-adrenoceptor-selective agonist)-induced tachycardia was significantly suppressed by ELF-EMF exposure in MF-1 for the increase in HR (DeltaHR), the decrease in QRS interval (DeltaQRS) and the decrease in QT (DeltaQT) interval. Adrenaline (nonselective beta-receptor agonist)-induced dysrhythmia was also significantly suppressed by ELF-EMF in MF-1 for the number of missing beats, the dysrhythmic ratio, and the increase in BP and PP. 5 These results indicated that 1-day exposure to ELF-EMF (60 Hz, 20 G) could suppress the increase in HR by affecting ventricular repolarization and may have a down-regulatory effect on responses of the cardiovascular system induced by sympathetic agonists.
Assuntos
Pressão Sanguínea/fisiologia , Campos Eletromagnéticos , Frequência Cardíaca/fisiologia , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Dobutamina/administração & dosagem , Dobutamina/farmacocinética , Relação Dose-Resposta a Droga , Regulação para Baixo , Eletrocardiografia/efeitos dos fármacos , Epinefrina/antagonistas & inibidores , Epinefrina/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hipotensão/induzido quimicamente , Injeções Intravenosas , Masculino , Ratos , Taquicardia/induzido quimicamente , Taquicardia/prevenção & controle , Fatores de TempoRESUMO
Esophageal reflux is a common condition that affects children and 1 in 10 adults, and if untreated may result in chronic esophagitis, aspiration pneumonia, esophageal strictures, and Barrett's esophagus, a premalignant condition. Although esophagitis is a multifactorial disease that may depend on transient lower esophageal sphincter (LES) relaxation, speed of esophageal clearance, mucosal resistance, and other factors, impairment of LES pressure is a common finding in patients complaining of chronic heartburn. Our data suggest that esophageal and LES circular muscle utilize distinct Ca2+ sources, phospholipid pools, and signal transduction pathways to contract in response to acetylcholine (ACh): (1) In esophageal muscle ACh-induced contraction requires influx of extracellular Ca2+ and may be linked to phosphatidylcholine metabolism, production of diacylglycerol (DAG) and arachidonic acid, and activation of a protein kinase C (PKC)-dependent pathway. (2) In LES muscle ACh-induced contraction utilizes intracellular Ca2+ release arising from metabolism of phosphatidylinositol (PI), and a calmodulin-myosin light chain kinase-dependent pathway. Resting LES tone, on the other hand, may be due to relatively low basal PI hydrolysis resulting in submaximal levels of inositol triphosphate (IP3)-induced calcium release and interaction with DAG to activate PKC. (3) After induction of experimental esophagitis, basal levels of PI hydrolysis and intracellular calcium stores are substantially reduced, resulting in a reduction of resting tone. In addition the signal transduction pathway responsible for LES contraction in response to ACh changes from one that depends on IP3 production, calcium release, and calmodulin activation to one that relies on influx of extracellular calcium and activation of PKC.
Assuntos
Esôfago/fisiologia , Transdução de Sinais , Acetilcolina/fisiologia , Esofagite/fisiopatologia , Junção Esofagogástrica/fisiologia , Humanos , Contração Muscular/fisiologiaRESUMO
1. The present study was aimed to investigate intracellular pathways involved in acetylcholine (ACh)-induced contraction in cat detrusor muscle cells 2. Contraction was expressed as per cent shortening of length of individually isolated smooth muscle cells obtained by enzymatic digestion. Dispersed intact and permeabilized cells were prepared for the treatment of drugs and antibody to enzymes, respectively. Using Western blot, we confirmed the presence of related proteins. 3. The maximal contraction to ACh was generated at 10(-11) M. This response was preferentially antagonized by M3 muscarinic receptor antagonist rho-fluoro-hexahydrosiladifenidol (rhoF-HSD) but not by the M1 antagonist pirenzepine and the M2 muscarinic receptor antagonist methoctramine. We identified G-proteins (Gq/11), (Gs), (G0), (Gi1), (Gi2) and (Gi3) in the bladder detrusor muscle. ACh-induced contraction was selectively inhibited by (Gq/11) antibody but not to other G subunit. 4. The phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor neomycin reduced ACh-induced contraction. However, the inhibitors of the phospholipase D, the phospholipase A2 and protein kinase C did not attenuate the ACh-induced contraction. ACh-induced contraction was inhibited by antibody to PLC-beta1 but not PLC-beta3 and PLC-gamma. Thapsigargin or strontium, which depletes or blocks intracellular calcium release, inhibited ACh-induced contraction. Inositol 1,4,5-triphosphate IP3 receptor inhibitor heparin reduced ACh-induced contraction. 5. These results suggest that in cat detrusor muscle contraction induced by ACh is mediated via M3 muscarinic receptor-dependent activation of Gq/11 and PLC-beta1 and IP3-dependent Ca(2+) release.
Assuntos
Acetilcolina/farmacologia , Músculo Liso/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Alcaloides , Animais , Benzofenantridinas , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/fisiologia , Gatos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas de Ligação ao GTP/metabolismo , Heparina/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Antagonistas Muscarínicos/farmacologia , Músculo Liso/citologia , Neomicina/farmacologia , Fenantridinas/farmacologia , Fosfolipases/antagonistas & inibidores , Fosfolipases/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Transdução de Sinais/fisiologia , Estrôncio/farmacologia , Tapsigargina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Bexiga Urinária/citologia , Ácido p-Cloromercurobenzoico/farmacologiaRESUMO
The involvement of non-adrenergic non-cholinergic (NANC) transmitters, such as adenosine 5'-triphosphate (ATP) and nitric oxide (NO), in the neurogenic relaxation of rat thoracic aorta was investigated in vessel segments suspended for isometric tension recording by polygraph. Responses to electrical field stimulation (EFS) and exogenous vasodilator were investigated in vessels precontracted with 5-hydroxytryptamine. EFS (100 V, 2-16 Hz, for 10 s at 3-min intervals), in the presence of guanethidine (10 microM) and atropine (10 microM) produced frequency-dependent relaxations. Pretreatment with tetrodotoxin (1 microM) markedly reduced the relaxation and desensitization with capsaicin (10 microM) significantly inhibited the relaxation. Exogenously added ATP caused concentration-dependent relaxations. Mechanical removal of the endothelium significantly inhibited EFS- and ATP-induced relaxation by 30+/-3% and 37+/-2%, respectively. Pretreatment with a P1-purinoceptor antagonist, 8-phenyltheophylline (10 microM) or P2X-purinoceptor antagonist, Evans blue (10 microM) did not influence the relaxations to EFS and exogenously added ATP. In contrast, the P2Y-purinoceptor antagonist, basilen blue (100 microM) markedly reduced the relaxations to EFS by 52+/-4% in the endothelium-intact preparations. However, in the endothelium-denuded preparations and capsaicin-pretreated preparations, basilen blue did not change relaxations elicited by EFS. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) also significantly inhibited the relaxations to EFS and ATP by 40+/-6% and 30+/-2%, respectively, in the endothelium-intact preparations but had no effect on the relaxations in the endothelium-denuded preparations or capsaicin-pretreated preparations. In addition, the EFS-induced relaxations were also inhibited 43+/-7% by pretreatment with 1H-[1,2,4]-oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 1 microM), soluble guanylate cyclase inhibitor. This study suggests that the NANC nerve system is present in the thoracic aorta of rat, mediating vasodilatation by sensory nerves. ATP, as a neurotransmitter released from sensory nerves, activates P2Y-purinoceptors located on the endothelium and stimulates the NO/cyclic GMP pathway, resulting in vasodilatation.
Assuntos
Aorta Torácica/fisiologia , Endotélio Vascular/fisiologia , Vasodilatação/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/inervação , Capsaicina/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Endotélio Vascular/citologia , Azul Evans/farmacologia , Técnicas In Vitro , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/fisiologia , Oxidiazóis/farmacologia , Antagonistas de Receptores Purinérgicos P1 , Antagonistas do Receptor Purinérgico P2 , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologia , Tetrodotoxina/farmacologia , Teofilina/análogos & derivados , Teofilina/farmacologia , Triazinas/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
The present study was designed to investigate the role of nitric oxide (NO), N-methyl-D-aspartate (NMDA) receptor and prostaglandins on hyperalgesia induced in rats by excitatory amino acids and the possibility that prostaglandins may act as the retrograde messenger in the spinal cord like NO. Nomega-nitro-L-arginine methyl ester (L-NAME; 500 microg/paw, intraplantarly (i.pl.)), MK-801 (10 microg/paw, i.pl.) or indomethacin (300 microg/paw, i.pl.) reduced the duration of phase 2 of the biting/licking and scratching (B/L + S) response induced by formalin injection from 255.6+/-16.7 s to 155.6+/-16.9, 172.25+/-33.3 or 205.6+/-16.7 s, respectively. L-NAME (0.3 mg, i.th.), MK-801 (8 microg, i.th.) or indomethacin (20 microg, i.th) reduced the duration of phase 2 of the B/L + S response induced by saline injection from 288.5+/-7.7s to 207.7+/-19.2, 184.6+/-7.7 or 1923+/-38.5 s, respectively. L-NAME or indomethacin injected into the spinal cord of the rat significantly reduced the hyperalgesia induced by NMDA (1 microg, i.th.) from 43.8+/-4.6% to 12.3+/-3.1 and 19.2+/-2.3%, respectively. It is assumed that NO produced by excitatory amino acids may increase prostaglandin production by cyclooxygenase activation. L-NAME, MK-801 or indomethacin injected into the rat spinal cord significantly reduced the hyperalgesia induced by prostaglandin E2 (PGE2, 25 ng, i.th.) in the tail-flick test from 40.6+/-3.5% to 18.2+/-3.2, 18.8+/-1.8 or 17.6+/-4.1%, respectively, but had little effect on hyperalgesia in the paw pressure test (except for indomethacin). In conclusion, NO and PGE2 affect the hyperalgesia induced by excitatory amino acids. It is suggested that PGE2, like NO, may act as a retrograde messenger in the spinal cord.
Assuntos
Dinoprostona/fisiologia , Aminoácidos Excitatórios/administração & dosagem , Hiperalgesia/fisiopatologia , Óxido Nítrico/fisiologia , Animais , Dinoprostona/administração & dosagem , Maleato de Dizocilpina/farmacologia , Formaldeído/administração & dosagem , Hiperalgesia/induzido quimicamente , Hiperalgesia/prevenção & controle , Indometacina/farmacologia , Masculino , N-Metilaspartato/administração & dosagem , NG-Nitroarginina Metil Éster/farmacologia , Dor/induzido quimicamente , Dor/fisiopatologia , Dor/prevenção & controle , Medição da Dor , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
1. We examined the role of the NO/cyclic GMP (cyclic GMP) pathway in nitric oxide (NO)- and vasoactive intestinal peptide (VIP)-induced relaxation of feline lower oesophageal sphincter (LES). Furthermore, it was studied whether methylene blue, LY83583 and ODQ, which are soluble guanylate cyclase (sGC) inhibitors, could inhibit NO-induced relaxation. 2. The nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine (L-NNA) had no effect in sodium nitropruside (SNP)-induced relaxation, but 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1)-induced relaxation was decreased by the pretreatment of L-NNA, which showed that SIN-1, not SNP, could activate NOS to cause relaxation. Methylene blue and LY83583 did not inhibit the relaxation by SNP and SIN-1. However, the more specific sGC inhibitor ODQ blocked the relaxation induced by NO donors. 3. To identify the relationship of NOS, sGC and adenylate cyclase in VIP-induced relaxation, tissue were pretreated with L-NNA and ODQ and SQ22536. These inhibitors produced significant inhibition of this response to VIP. The adenylyl cyclase inhibitor SQ 22536 also inhibited relaxation by VIP. 4. In conclusion, our data showed that SNP- and SIN-1-induced relaxation was mediated by sGC. Of sGC inhibitors, methylene blue and LY83583 were not adequate for the examination of NO donor-induced feline LES smooth muscle relaxation. VIP also caused relaxation by the pathway involving NO and cGMP and cAMP.
Assuntos
GMP Cíclico/fisiologia , Junção Esofagogástrica/fisiologia , Óxido Nítrico/fisiologia , Transdução de Sinais/fisiologia , Aminoquinolinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Gatos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Junção Esofagogástrica/efeitos dos fármacos , Junção Esofagogástrica/inervação , Técnicas In Vitro , Indometacina/farmacologia , Masculino , Azul de Metileno/farmacologia , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroprussiato/farmacologia , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
1. This study was aimed at evaluating the effect of rutin and harmaline (1-methyl-7-methoxy-3,4-dihydro-beta-carboline) on the development of the surgically induced reflux oesophagitis, on gastric secretion, lipid peroxidation, polymorphonucleocytes (PMNs) accumulation, superoxide and hydroxyl radical production in PMNs, cytokine [interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha)] production in blood and [Ca2+]i mobilization in PMNs. 2. Rutin and harmaline significantly prevented the development of reflux oesophagitis and gastric secretion. Treatments of oesophagitis rats with rutin and harmaline inhibited lipid peroxidation, and myeloperoxidase (MPO) in the oesophagus in comparison with untreated rats. 3. Superoxide anion and hydrogen peroxide production in 1 microm formylmethionylleucylphenylalanine (fMLP)- or 0.1 microg ml-1N-phorbol 12-myristate 13-acetate (PMA)-activated PMNs was inhibited by rutin and harmaline in a dose-dependent fashion. Rutin and harmaline effectively scavenged the hydroxyl radical and hydrogen peroxide. Treatments of oesophagitis rats with rutin and harmaline inhibited IL-1beta production in the oesophagus in comparison with untreated rats, but TNF-alpha production was not affected by rutin and harmaline. The fMLP-induced elevation of [Ca2+]i was inhibited by rutin. 4. The results of this study suggest that rutin and harmaline may have beneficial protective effects against reflux oesophagitis by the inhibition of gastric acid secretion, oxidative stress, inflammatory cytokine production (i.e. IL-1beta), and intracellular calcium mobilization in PMNs in rats.
Assuntos
Esofagite Péptica/prevenção & controle , Harmalina/uso terapêutico , Rutina/uso terapêutico , Animais , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Esofagite Péptica/metabolismo , Esôfago/enzimologia , Ácido Gástrico/metabolismo , Harmalina/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Peroxidase/biossíntese , Ratos , Ratos Sprague-Dawley , Rutina/farmacologiaRESUMO
1. In the present investigation we examined the regulation of calmodulin (CaM)- and protein kinase C (PKC)-dependent pathways by cytosolic Ca(2+) in the contraction of cat lower oesophageal sphincter (LES). 2. Force developed in response to increasing doses of acetylcholine (ACh) was directly related to the increase of the [Ca(2+)](i) measured by fura-2. Thapsigargin, which depletes Ca(2+) stores, reduced the contraction and the [Ca(2+)](i). In addition, contraction in response to maximal ACh was reduced by the CaM inhibitor CGS9343B but not by the PKC inhibitor chelerythrine. The contraction in response to submaximal ACh was reduced by chelerythrine but not by CGS9343B. 3. In permeabilized cells, the contraction in response to low Ca(2+) (0.54 microm) was also reduced by CGS9343B. 4. The response to high Ca(2+) (1.0 microm) was reduced by CGS9343B. ACh also inhibited PKC activation induced by diacylglycerol, which activation is inhibited by the N-myristoylated peptide inhibitor derived from pseudosubstrate sequences of PKCalphabetagamma (myr-PKC-alphabetagamma), but not of myr-PKC-alpha. 5. These data are consistent with the view that activated CaM-dependent pathways inhibit PKC-dependent pathways, this switch mechanism might be regulated by Ca(2+) in the LES.
Assuntos
Cálcio/fisiologia , Calmodulina/fisiologia , Junção Esofagogástrica/fisiologia , Proteína Quinase C/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Acetilcolina/antagonistas & inibidores , Acetilcolina/farmacologia , Alcaloides , Animais , Benzimidazóis/farmacologia , Benzofenantridinas , Cálcio/antagonistas & inibidores , Cálcio/química , Calmodulina/química , Gatos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Diglicerídeos/farmacologia , Junção Esofagogástrica/citologia , Junção Esofagogástrica/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fenantridinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Transdução de Sinais/fisiologia , Tapsigargina/antagonistas & inibidores , Tapsigargina/farmacologiaRESUMO
We have previously shown that, in circular muscle cells of the lower esophageal sphincter (LES) isolated by enzymatic digestion, contraction in response to maximally effective doses of acetylcholine (ACh) or Inositol Triphosphate (IP3) depends on the release of Ca2+ from intracellular stores and activation of a Ca2+-calmodulin (CaM)-dependent pathway. On the contrary, maintenance of LES tone, and response to low doses of ACh or IP3 depend on a protein kinase C (PKC) mediated pathway. In the present investigation, we have examined requirements for Ca2+ regulation of the interaction between CaM- and PKC-dependent pathways in LES contraction. Thapsigargin (TG) treatment for 30 min dose dependently reduced ACh-induced contraction of permeable LES cells in free Ca2+ medium. ACh-induced contraction following the low level of reduction of Ca2+ stores by a low dose of TG (10(-9) M) was blocked by the CaM antagonist, CGS9343B but not by the PKC antagonists chelerythrine or H7, indicating that the contraction is CaM-dependent. After maximal reduction in intracellular Ca2+ from Ca2+ stores by TG (10(-6) M), ACh-induced contraction was blocked by chelerythrine or H7, but not by CGS9343B, indicating that it is PKC-dependent. In normal Ca2+ medium, the contraction by ACh after TG (10(-9) M) treatment was also CaM-dependent, whereas the contraction by ACh after TG (10(-9) M) treatment was PKC-dependent. We examined whether PKC activation was inhibited by activated CaM. CGS 9343B inhibited the CaM-induced contraction, but did not inhibit the DAG-induced contraction. CaM inhibited the DAG-induced contraction in the presence of CGS 9343B. This inhibition by CaM was Ca2+ dependent. These data are consistent with the view that the switch from a PKC-dependent pathway to a CaM dependent pathway can occur and can be regulated by cytosolic Ca2+ in the LES.
Assuntos
Calmodulina/farmacologia , Junção Esofagogástrica/fisiologia , Contração Muscular/efeitos dos fármacos , Proteína Quinase C/fisiologia , Acetilcolina/farmacologia , Animais , Cálcio/farmacologia , Gatos , Feminino , Técnicas In Vitro , Masculino , Tapsigargina/farmacologiaRESUMO
Endothelin-1 (ET-1), a novel and potent vasoconstrictor in blood vessel, is known to have some functions in the rat central nervous system (CNS). In order to investigate the central functions of ET-1, ET-1 was administered to the periaqueductal gray area (PAG) of anesthetized rats to induce barrel rolling and increase the arterial blood pressure (ABP). ET-1 had a modulatory effect on central cardiovascular and behavioral control. The selective N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (3 micromol/kg, i.p.) blocked the ET-1 induced responses, and both the nitric oxide synthase (NOS) inhibitor L-NAME (N-nitro-L-arginine methylester 1 mmol/rat) and the nitric oxide (NO) scavenger hemoglobin (15 nmol/rat) had similar effects in reducing the ET-1 (10 pmol/rat)-induced behavioral changes and ABP elevation. However, NO donor sodium nitroprusside (SNP 10 microg, 1 microg/rat) decreased the ET-1 induced ABP elevation, and recovered the ET-1-induced barrel rolling effect that was reduced by MK-801. These results suggest that ET-1 might have neuromodulatory functions such as ABP elevation and barrel rolling induction in the PAG of the rats via the NMDA receptor and NO.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Endotelina-1/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/fisiologia , Sistema Cardiovascular , Maleato de Dizocilpina/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Frequência Cardíaca/fisiologia , Masculino , Atividade Motora/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Nitroprussiato/farmacologia , Substância Cinzenta Periaquedutal/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
1. In normal rats, intracerebroventricular (i.c.v.) guanabenz induced a decrease in blood pressure (BP) and heart rate (HR), and this hypotension or bradycardia was not changed by rANF pretreatment (3 micrograms i.c.v.). 2. In pithed rats, intravenous (i.v.) guanabenz, an alpha 2-adrenoceptor agonist, produced an increase in mean blood pressure (MBP) in a dose-dependent manner. The pressor response by guanabenz was attenuated by infusion of rANF. This attenuation was additive when incubated in combination with yohimbine. 3. In pithed rats, the pressor response due to the increase of sympathetic outflow with electrical stimulation was partially blocked by rANF infusion or chronic guanabenz treatment. This reduction was not augmented by chronic guanabenz plus rANF treatment. 4. The inhibitory action of guanabenz in tachycardia evoked by electrical stimulation at the C7-T1 site was attenuated by rANF, but not by chronic treatment with guanabenz.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Fator Natriurético Atrial/farmacologia , Estado de Descerebração/fisiopatologia , Guanabenzo/farmacologia , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores Pré-Sinápticos/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/administração & dosagem , Animais , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Guanabenzo/administração & dosagem , Frequência Cardíaca/efeitos dos fármacos , Masculino , Fenilefrina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 2/fisiologia , Receptores Pré-Sinápticos/fisiologiaRESUMO
1. This study investigates possible mechanisms through which imipramine (IMI) exerts its antienuretic effect. The micturition reflex in response to bladder distension produced by saline infusion was examined in anaesthetized rats. 2. The amplitude and frequency of micturition reflex contractions were reduced by peripheral administration of IMI, but the micturition reflex was abolished after its intracerebroventricular (i.c.v.) administration. A muscarinic antagonist, atropine, displayed an inhibitory effect similar to that of IMI. A muscarinic agonist, carbachol, produced a dose-related rightward shift of the dose-response curve to IMI. Both IMI i.c.v. and the muscarinic antagonist l-methylscopolamine i.c.v. elevated the threshold of volume and pressure for micturition initiation, indicating that IMI and muscarinic antagonists mainly exert a central inhibitory effect on the micturition reflex. 3. In addition, to evaluate the role of central monoaminergic neurotransmission on micturition, the acetylcholine depletor hemicholinium-3 (HC-3), the catecholamines depletor alpha-methyl-p-tyrosine (AMPT), and the serotonin depletor p-chlorophenylalanine (PCPA) were examined alone or in combination with IMI. The micturition threshold was increased by treatment with HC-3, but not by AMPT or PCPA. In HC-3 treated rats, the inhibitory effect of IMI on the micturition reflex was more prolonged than in normal rats. After administration of IMI, the recovery from the cessation of micturition reflex contractions was facilitated by carbachol in normal rats, but not in HC-3 treated rats. This indicates that acetylcholine plays a facilitatory role in initiating micturition reflex contractions. 4. Acute treatment with IMI decreased the frequency and increased the volume threshold of micturition reflex contraction. Acute and chronic treatment with IMI prolonged the cessation period of micturition by IMI. 5. These results suggest that IMI exerts an inhibitory action on the micturition reflex by a central cholinergic mechanism. Muscarinic receptors located at the supraspinal level are tonically stimulated during distension-induced micturition reflex.
Assuntos
Inibidores da Captação Adrenérgica/farmacologia , Antidepressivos Tricíclicos/farmacologia , Enurese/tratamento farmacológico , Imipramina/farmacologia , Micção/efeitos dos fármacos , Inibidores da Captação Adrenérgica/administração & dosagem , Inibidores da Captação Adrenérgica/uso terapêutico , Animais , Antidepressivos Tricíclicos/administração & dosagem , Antidepressivos Tricíclicos/uso terapêutico , Atropina/administração & dosagem , Atropina/farmacologia , Atropina/uso terapêutico , Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Feminino , Fenclonina/farmacologia , Hemicolínio 3/farmacologia , Imipramina/administração & dosagem , Imipramina/uso terapêutico , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Intraventriculares , Metiltirosinas/farmacologia , Antagonistas Muscarínicos/administração & dosagem , Antagonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/uso terapêutico , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , N-Metilescopolamina , Parassimpatolíticos/administração & dosagem , Parassimpatolíticos/farmacologia , Ratos , Ratos Sprague-Dawley , Derivados da Escopolamina/administração & dosagem , Derivados da Escopolamina/farmacologia , Serotoninérgicos/farmacologia , alfa-MetiltirosinaRESUMO
Acetylcholine (ACh)-induced contraction of esophageal circular smooth muscle cells was inhibited by the M2 muscarinic antagonist methoctramine. In lower esophageal sphincter (LES) cells contraction was inhibited by the M3 antagonist p-fluoro-hexa-hydro-sila-difenidol (pF-HSD). Pertussis toxin (PTX) reduced ACh-induced contraction of esophageal but not of LES cells, which suggested that different receptor-linked G proteins are involved. Antibodies against G13 antagonized contraction of esophageal cells and G9-G11 antibodies antagonized contraction of LES cells. The phosphatidylinositol-specific phospholipase C (PLC) inhibitors, U-73122 and neomycin, reduced ACh-induced contraction of LES but not of esophageal cells. Conversely, propranolol and p-chloromercuribenzoic acid (pCMB), which inhibit a phosphatidylcholine-specific phospholipase D (PLD)-dependent pathway, reduced contraction of esophageal but not of LES muscle cells. At 1 and 5 sec after the administration of ACh (10(-5) M), inositol 1,4,5-trisphosphate (IP3) increased only in LES muscle, which suggested that contraction results from PLC-induced IP3 production in the LES but not in the esophagus. The IP3 receptor antagonist heparin, and depletion of intracellular Ca++ stores by thapsigargin or A23187, inhibited ACh-induced contraction of LES but not of esophageal muscle. It was concluded that ACh-induced esophageal contraction depends preferentially on M2 receptors, a PTX-sensitive G13 protein, phosphatidylcholine-specific PLD and production of diacylglycerol (DAG) and is independent of IP3 formation and the release of intracellular Ca++. Conversely, LES contraction is mediated through M3 receptors, a PTX-insensitive G9-G11 protein, activation of PLC, IP3 formation and the release of intracellular Ca++.
Assuntos
Junção Esofagogástrica/fisiologia , Esôfago/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Fosfolipases/metabolismo , Receptores Muscarínicos/fisiologia , Transdução de Sinais/fisiologia , Acetilcolina/farmacologia , Animais , Gatos , Diglicerídeos/biossíntese , Ativação Enzimática/efeitos dos fármacos , Junção Esofagogástrica/metabolismo , Junção Esofagogástrica/ultraestrutura , Esôfago/metabolismo , Esôfago/ultraestrutura , Feminino , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/química , Músculo Liso/enzimologia , Toxina Pertussis , Fosfatidilinositóis/metabolismo , Fosfolipase D/metabolismo , Sensibilidade e Especificidade , Fosfolipases Tipo C/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
We have shown that acetylcholine (ACh)-induced contraction of esophageal circular muscle cells is mediated by activation of protein kinase C (PKC). We now examine the role of phospholipase A2 (PLA2). ACh increases [3H]arachidonic acid release in esophageal but not in lower esophageal sphincter (LES) muscle. In addition, ACh-induced contraction of esophageal but not of LES cells was reduced by the PLA2 antagonist dimethyleicosadienoic acid and by antiserum to a 100-kDa cytosolic PLA2 (cPLA2). These data suggest that the 100-kDa cPLA2 plays a role in ACh-induced contraction of esophageal but not of LES muscle. In esophageal cells, arachidonic acid produced by PLA2 caused little contraction by itself but potentiated contraction induced by the PKC agonist diacylglycerol (DAG). The free fatty acids linoleic acid and linolenic acid also potentiated DAG-induced contraction. Indomethacin and nordihydroguaiaretic acid had no effect on arachidonic acid-induced potentiation of DAG. The potentiation of DAG-induced contraction by arachidonic acid was inhibited by the PKC inhibitor H-7, but it was not affected by the calmodulin inhibitor CGS-9343B. We conclude that a 100-kDa cPLA2 participates in ACh-induced esophageal contraction by producing arachidonic acid and potentiating DAG-induced activation of a PKC-dependent pathway.
Assuntos
Acetilcolina/farmacologia , Citosol/enzimologia , Esôfago/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Fosfolipases A/fisiologia , Animais , Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/farmacologia , Gatos , Diglicerídeos/farmacologia , Sinergismo Farmacológico , Junção Esofagogástrica/citologia , Junção Esofagogástrica/efeitos dos fármacos , Junção Esofagogástrica/fisiologia , Esôfago/citologia , Esôfago/fisiologia , Feminino , Masculino , Peso Molecular , Músculo Liso/citologia , Músculo Liso/fisiologia , Fosfolipases A/metabolismo , Fosfolipases A2 , Testes de PrecipitinaRESUMO
Smooth muscle cells isolated from the circular muscle layer of cat esophagus and lower esophageal sphincter (LES) exhibit distinct contractile intracellular signal transduction pathways in response to acetylcholine. To determine whether these contractile pathways are muscle type dependent, the authors examined the signal transduction pathways utilized by substance P and bombesin, which in other tissues, use different signal transduction pathways, and by the GTP analog, guanosine 5'-O-3-thiotriphosphate (GTP gamma S), which activates all available G proteins. Western blot analysis of esophageal and LES circular muscle revealed the presence of Gq-G11 (42 kD), Gi1-Gi2 (40 kD) and Go-Gi3 (40 kD) types of G proteins. The responses of esophageal cells to bombesin and substance P were blocked by 1) a Gi3 protein antibody, 2) the inhibitor of specific phosphatidylcholine-phospholipase C (PLC) D609 potassium tricyclo-[5.2.1.0(2.6)]-decyl-(9[8])-xanthogenate, 3) inhibition of phosphatidic acid phosphohydrolase by propranolol, 4) the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and 5) incubation in Ca(++)-free medium. Conversely, the responses of LES muscle cells to bombesin and substance P were blocked by 1) a Gq-G11 antibody, 2) a phosphatidylinositol-specific PLC antagonist U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), 3) the calmodulin inhibitor CGS9343B (1,3-Dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin++ +-4 - yl)methyl-4-piperindinyl]-2H-benzimidazol-2-one maleate) and 4) incubation in Sr++. After permeabilization by saponin, inositol 1,4,5-trisphosphate contracted LES but not esophageal cells. The inositol 1,4,5-trisphosphate receptor antagonist heparin and depletion of intracellular Ca++ stores by thapsigargin or A23187 4-Benzoxazolecarboxylic acid, 5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol- 2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-, [6s-[6.alpha. (2S*,3S*),8.beta. (R*), 9.beta., 11. alpha.]]-(9Cl), blocked bombesin- and substance P-induced contraction of LES but not of esophageal muscle. In addition, contraction in response to GTP gamma S, which activates all G proteins, was blocked in esophageal cells by a Gi3-protein antibody, propranolol, D609 and H7. In LES muscle cells, the response to GTP gamma S was blocked by a Gq protein antibody, U-73122 and CGS934B. These data demonstrate that, in esophageal muscle, different agonists activate the same Gi3 protein, phosphatidylcholine-specific phospholipases and protein kinase C-dependent pathway.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Bombesina/farmacologia , Junção Esofagogástrica/metabolismo , Esôfago/metabolismo , Músculo Liso/metabolismo , Transdução de Sinais , Substância P/farmacologia , Animais , Gatos , Feminino , Proteínas de Ligação ao GTP/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Fosfolipases Tipo C/fisiologiaRESUMO
We previously showed that lower esophageal spincter (LES) tone depends on spontaneous production of inositol 1,4,5-trisphosphate (IP3) and release of intracellular Ca2+ and that acute experimental esophagitis reduces LES tone and IP3 production, suggesting damage to mechanisms responsible for release of Ca2+ from intracellular stores. In the present investigation, we examined the possibility that mechanisms responsible for Ca2+ storage or uptake may also be damaged. LES circular muscle cells were isolated by enzymatic digestion. Contraction was measured in response to IP3 and thapsigargin, which enhances release of Ca2+ from intracellular stores, and in response to calmodulin and to diacylglycerol. In addition, normal cells were incubated in thapsigargin to assess the effect of depletion of intracellular Ca2+ stores on contractile response. Contraction in response to IP3 and thapsigargin was reduced in experimental esophagitis, but contraction in response to calmodulin or diacylglycerol was not. Acetylcholine (ACh)-induced contraction of normal cells was inhibited by the calmodulin antagonist CGS-9343B but not by 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7). In contrast, in cells from animals with esophagitis or in thapsigargin-treated cells from normal animals, ACh-induced contraction was inhibited by H-7 and not by CGS-9343B. We conclude that experimental esophagitis may damage intracellular Ca2+ stores in the LES and change the intracellular contractile pathways activated by ACh from calmodulin dependent in normal cells to protein kinase C dependent in esophagitis.
Assuntos
Cálcio/metabolismo , Esofagite/fisiopatologia , Junção Esofagogástrica/fisiopatologia , Músculo Liso/fisiopatologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Acetilcolina/farmacologia , Análise de Variância , Animais , Benzimidazóis/farmacologia , Calmodulina/antagonistas & inibidores , Calmodulina/farmacologia , Gatos , Diglicerídeos/farmacologia , Junção Esofagogástrica/efeitos dos fármacos , Junção Esofagogástrica/fisiologia , Feminino , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/farmacologia , Cinética , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Tapsigargina/farmacologia , Fatores de TempoRESUMO
BACKGROUND & AIMS: In esophageal circular muscle, acetylcholine activates phosphatidylcholine-specific phospholipases C and D and phospholipase A2, producing diacylglycerol and arachidonic acid, which cause contraction by interacting synergistically to activate protein kinase C. In a model of acute esophagitis, leukotriene D4 (LTD4) contributes to acetylcholine-induced contraction. We examined intracellular signaling in LTD4-induced contraction. METHODS: Esophageal and lower esophageal sphincter (LES) cells, isolated by enzymatic digestion, were contracted by LTD4 in the absence or presence of inhibitors. Permeabilization by saponin allowed use of G-protein antibodies and heparin. RESULTS: Esophageal contraction was inhibited by pertussis toxin, Gi3 antibodies, D609 (phosphatidylcholine-specific phospholipase C inhibitor), propranolol (phospholipase D pathway inhibitor), and chelerythrine (protein kinase C antagonist) but not W7 (calmodulin antagonist). LES contraction was unaffected by pertussis toxin. It was inhibited by Gq antibodies, U-73122 (phosphatidylinositol-specific phospholipase C inhibitor), heparin (inositol 1,4,5-trisphosphate inhibitor), and W7 and reduced by D609. CONCLUSIONS: In the esophagus, LTD4 activates a protein kinase C-dependent pathway through pertussis toxin-sensitive Gi3 proteins and phosphatidylcholine-specific phospholipase. In the LES, LTD4 activates a calmodulin-dependent pathway through pertussis toxin-insensitive Gq proteins and phosphatidylinositol-specific phospholipase C. The intracellular pathways activated by LTD4 in the esophagus and the LES are similar to those activated by acetylcholine and other agonists.
Assuntos
Junção Esofagogástrica/efeitos dos fármacos , Esôfago/efeitos dos fármacos , Leucotrieno D4/farmacologia , Contração Muscular , Músculo Liso/efeitos dos fármacos , Animais , Calmodulina/fisiologia , Gatos , Feminino , Proteínas de Ligação ao GTP/fisiologia , Masculino , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Fosfatidilinositóis/fisiologia , Fosfolipases/fisiologia , Proteína Quinase C/fisiologiaRESUMO
Esophageal circular muscle cells isolated by enzymatic digestion contracted in response to acetylcholine (ACh) and in response to the protein kinase C (PKC) agonist 1,2-dioctanoylglycerol (1,2 DAG). Both responses were blocked by PKC antagonists but not by calmodulin antagonists. Furthermore, specific PKC activity, measured in the particulate fraction of the muscle, increased in response to cholinergic stimulation, suggesting that ACh-induced contraction is mediated by a PKC-dependent pathway. ACh-induced contraction decreased with decreasing extracellular Ca2+ and was blocked in Ca(2+)-free physiological salt solution (PSS). Similarly, contraction by the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) was blocked by removal of Ca2+ from the PSS. Diacylglycerol production in response to ACh was reduced when extracellular Ca2+ was reduced from 2 to 0.5 mM and was abolished in Ca(2+)-free PSS. The response to 1,2-DAG, however, did not significantly change as extracellular Ca2+ or cytosolic Ca2+ was reduced to zero. Heparin (10 micrograms/ml), thapsigargin (3 microM), or the Ca2+ ionophore A-23187 (3 microM) had no effect on 1,2-DAG or ACh-induced contraction in permeable cells. The data suggest that contraction in response to ACh is mediated by influx of extracellular Ca2+ and a PKC-dependent pathway. Ca2+ may be required mainly to activate the phospholipases responsible for production of diacylglycerol, since contraction of esophageal muscle cells in response to 1,2-DAG is Ca2+ independent.