Progesterone inhibits gallbladder motility through multiple signaling pathways

Progesterone (P) has an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since P levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of P and two metabolites on contraction in guinea pig gallbladder strips. P induced a concentration-dependent relaxation in guinea pig gallbladder strips precontracted with cholecystokinin octapeptide (CCK). Pretreatment of gallbladder strips with P (50 microM) also reduced the amount of CCK-induced tension. Nifedipine (1 microM) produced a similar effect. Pretreatment of the strips with PKA inhibitor 14--22 amide myristolated (180 nM) or the PKG inhibitor KT5823 (1.2 microM) either separately or in combination significantly reduced the amount of P-induced relaxation. Rp-cAMPs (0.1mM) or H-89 (10 microM) separately or in combination significantly reduced the P-effect; however, the combination of agents produced the largest reduction. Genistein (1 microM), an inhibitor of protein tyrosine kinases, significantly (p<0.01) reduced the amount of P-induced relaxation. The use of strontium in the Kreb's solution as a substitute for Ca(2+) significantly (p<0.01) reduced the amount of CCK-induced tension. Pretreatment of the strips with 2-APB (26 microM), an inhibitor of IP(3,) induced Ca(2+) release, produced a significant (p<0.01) reduction in P-induced relaxation. We conclude that P inhibits gallbladder motility rapidly by nongenomic actions of the hormone. Several pathways that include tyrosine kinase and PKA/cAMP activity may mediate this effect.     

17β-Estradiol relaxes cholecystokinin- and KCl-induced tension in male guinea pig gallbladder strips

Estrogen has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since estrogen and progesterone levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17β-estradiol (E2) on contraction in male guinea pig gallbladder strips. E2 induced a concentration-dependent relaxation of either CCK-induced tension or KCl-induced tension. Pretreatment of the strips with PKA inhibitor 14-22 amide myristolated had no significant effect on the E2-induced relaxation. Pretreatment of strips with 2-APB, and inhibitor of IP₃ induced Ca²⁺ release, produced a significant (p < 0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used to induce tension. KT5823, an inhibitor of PKG, also significantly (p < 0.001) increased the amount of E2-induced relaxation. Genistein, an inhibitor of protein tyrosine kinase, had no significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl- when used in combination had no significant effect on the amount of CCK-induced tension, but significantly (p < 0.001) increased the amount of E2-induced relaxation. When E2 was added to the chambers prior to either CCK or KCl, a significant decrease (p < 0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca²⁺ entry mediates the E2-induced relaxation of CCK- and KCl-induced tension in male guinea pig gallbladder strips.     

Biochemical Evidence for Alteration of Neostriatal Dopaminergic Function by 5,7-Dihydroxytryptamine

Unilateral injection of 5,7-dihydroxytryptamine (DHT) into the rat neostriatum markedly reduced not only striatal tryptophan hydroxylase (TPH) activity but also striatal tyrosine hydroxylase (TH) activity and dopamine (DA) concentration measured 10--15 days later. The decrease in striatal TH activity was dose related over the range of 8--32 micrograms of DHT; a dose of 16 micrograms reduced striatal TH activity to 40--50% of control, DA concentration to 38% of control, and TPH activity to 5--20% of control. Intrastriatal injection of 16 micrograms of DHT reduced TH activity in the ipsilateral substantia nigra to 51% of control. Pretreatment with amfonelic acid, a potent DA uptake inhibitor, significantly reduced the effect of DHT on striatal and nigral TH activity and striatal DA concentration without affecting the DHT-induced decrease in striatal TPH activity. Desmethylimipramine (5 and 25 mg/kg) had no effect on the DHT-induced decrease in striatal TH activity. Striatal choline acetyltransferase and glutamic acid decarboxylase activities were not decreased by 16 micrograms of DHT. The results indicate that DHT can alter dopaminergic function in the rat neostriatum through a direct effect of the drug on DA neurons.     

Differential regulation of gonadotropin-releasing hormone secretion and gene expression by androgen: membrane versus nuclear receptor activation

Steroid hormones induce rapid membrane receptor-mediated effects that appear to be separate from long-term genomic events. The membrane receptor-mediated effects of androgens on GT1-7 GnRH-secreting neurons were examined. We observed androgen binding activity with a cell-impermeable BSA-conjugated testosterone [testosterone 3-(O-carboxymethyl)oxime (T-3-BSA)] and were able to detect a 110-kDa protein recognized by the androgen receptor (AR) monoclonal MA1-150 antibody in the plasma membrane fraction of the GT1-7 cells by Western analysis. Further, a transfected green fluorescent protein-tagged AR translocates and colocalizes to the plasma membrane of the GT1-7 neuron. Treatment with 10 nM 5alpha-dihydrotestosterone (DHT) inhibits forskolin-stimulated accumulation of cAMP, through a pertussis toxin-sensitive G protein, but has no effect on basal cAMP levels. The inhibition of forskolin-stimulated cAMP accumulation by DHT was blocked by hydroxyflutamide, a specific inhibitor of the nuclear AR. DHT, testosterone (T), and T-3-BSA, all caused significant elevations in intracellular calcium concentrations ([Ca(2+)](i)). T-3-BSA stimulates GnRH secretion 2-fold in the GT1-7 neuron, as did DHT or T. Interestingly GnRH mRNA levels were down-regulated by DHT and T as has been reported, but not by treatment with T-3-BSA or testosterone 17beta-hemisuccinate BSA. These studies indicate that androgen can differentially regulate GnRH secretion and gene expression through specific membrane-mediated or nuclear mechanisms.     

Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro

This study investigates the involvement of capacitative Ca2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM-1 microM, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 microM) after 55 min. Contractile responses to thapsigargin (1 microM) were abolished in a Ca(2+)-free medium. Subsequent re-addition of Ca2+ (2.5 mM) produced a sustained tonic contraction (99 +/- 6% of the carbachol response). The contractile response to Ca2+ re-addition following incubation of tissues in a Ca(2+)-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 +/- 4 % vs 100 +/- 7 % of carbachol; p < 0.05). Contractile responses to Ca2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2+ channel antagonist, nifedipine (10 microM) and (b) the general inhibitor of Ca2+ entry channels including store-operated channels, SK&F96365 (50 microM and 100 microM). In separate experiments, responses to Ca2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 microM). These results suggest that capacitative Ca2+ entry provides a source of activator Ca2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2+ re-addition following depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin, are mediated in part by Ca2+ entry through voltage-operated Ca2+ channels and by capacitative Ca2+ entry through store-operated Ca2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2+ entry in this tissue may be modulated by tyrosine kinase.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.     

Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.     

八肽胆囊收缩素激活钙离子通道和酪氨酸激酶诱导豚鼠心肌细胞内的游离钙增高

探讨八肽胆囊收缩素(CCK-8)对豚鼠单个心肌细胞内游离钙浓度([Ca2+]i的影响及其信号转导机制.Fluo 3-AM标记酶消化法分离的单个心室肌细胞,用激光共聚焦显微镜测定细胞内[Ca2+]i的浓度.[Ca2+]i的变化用荧光强度(Fi)和相对荧光强度(Fi/F0%)表示.实验结果如下(1)在含Ca2+1.0 mmol/L的Tyrode's液中,CCK-8(1～104pmoVL)均可引起[Ca2+]i快速显著上升(P＜0.01).(2)用钙离子鳌合剂EGTA(3 mmol/L)和钙离子通道阻断剂nisoldipine(0.5μmol/L)预孵育心肌细胞5 min,CCK-8(102pmol/L)仅可引起[Ca2+]i缓慢轻度上升(P＜0.01).(3)用非选择性CCK受体拮抗剂丙谷胺(proglumide 6μmo1/L)或酪氨酸激酶抑制剂genistein(1 μmol/L)预孵育心肌细胞5 min,则完全抑制CCK-8诱导的[Ca2+]i升高(P＜0.01).CCK-8可通过激活其受体控制的Ca2+通道,引起Ca2+内流,诱导细胞内Ca2+释放,引起豚鼠单个心肌细胞内[Ca2+]i上升,此作用可能由酪氨酸激酶介导.     

Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.     

Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca(2+) release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca(2+) release and CaM kinase II activity.     

Androgens and the immunocompetence handicap hypothesis: unraveling direct and indirect pathways of immunosuppression in song sparrows

The immunocompetence handicap hypothesis proposes that testosterone (T)-dependent sexual signals are honest indicators of male health or genetic quality because only high-quality males are able to withstand the obligate effects of T-induced immunosuppression. In birds, the basic assumption that T suppresses immune function is equivocal, and the physiological mechanisms underlying T-induced immunosuppression remain to be investigated. We explored the proximate pathways of T-induced immunosuppression in song sparrows (Melospiza melodia) by treating captive nonbreeding males with different androgens and measuring several components of acquired immune function. Males implanted with T suppressed cell-mediated and humoral immune responses compared to males implanted with 5alpha-dihydrotestosterone (DHT), dehydroepiandrosterone, or control (empty) implants. Furthermore, T treatment increased plasma levels of corticosterone and decreased body mass and fat stores in relation to other treatments. The failure of DHT to depress immune function suggests that T-induced immunosuppression does not occur through a direct pathway because both T and DHT bind to androgen receptors on target cells. Instead, we outline indirect pathways that are likely responsible for suppression of the avian immune system that include stress-induced immunosuppression, aromatization to estrogen, and alterations in energy allocation that constrain expenditures toward immune system activation.     

Maintenance of resting tension in the american eel (Anguilla rostrata L.) heart is dependent upon exogenous fuel and the sarcoplasmic reticulum

The relationship between extracellular glucose and management of cell Ca(2+) in the heart of the American eel (Anguilla rostrata) was indirectly assessed by monitoring the performance of isolated ventricular strips at 20 degrees C. Twitch force increased in ventricular strips under specific conditions of 30 bpm pacing and an extracellular Ca(2+) challenge from 1.5 to 9.5 mM. The response was independent of any exogenous metabolic fuel in the medium. Resting tension was maintained when glucose was available, but in the absence of a metabolic fuel, resting tension increased in response to the increase in extracellular Ca(2+) level. When ventricular strips were treated with iodoacetate to inhibit glycolysis, a Ca(2+) challenge resulted in a decrease in twitch force in association with an approximately equivalent increase in resting tension even in the presence of exogenous glucose. However, when pyruvate (5 mM) was substituted as a metabolic fuel, twitch force increased as a function of extracellular Ca(2+), and resting tension was maintained in the presence of iodoacetate. Therefore, there is a need for an extracellular fuel but not a specific metabolic requirement for glucose to maintain the performance characteristics, which are presumably related to the management of intracellular Ca(2+) levels. Ventricular strips were treated with ryanodine to inhibit Ca(2+) release and uptake by the sarcoplasmic reticulum (SR). Ryanodine treatment impaired postrest potentiation at high extracellular Ca(2+) levels. In the presence of ryanodine, the protective effect of glucose on the increase in resting tension in the face of an extracellular Ca(2+) challenge was eliminated. Considered together, the results reveal that the heart of the American eel has a requirement for an extracellular fuel to manage intracellular Ca(2+) at high Ca(2+) loads, and that the SR plays a role in the beat-to-beat regulation of Ca(2+) at a frequency of 30 bpm, high Ca(2+) load, and 20 degrees C.     

Effects of bile acids on the muscle functions of guinea pig gallbladder

Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused H2O2 production, lipid peroxidation, and increased PGE2 synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase A2 (cPLA2), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of H2O2 via activation of NADPH and xanthine oxidase. H2O2 causes lipid peroxidation and activates cPLA2 to increase PGE2 production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway.     

Genistein inhibits Ca2+ influx and glutamate release from hippocampal synaptosomes: putative non-specific effects

The role of protein tyrosine kinases on glutamate release was investigated by determining the effect of broad range inhibitors of tyrosine kinases on the release of glutamate from rat hippocampal synaptosomes. We found that lavendustin A and herbimycin A did not inhibit glutamate release stimulated by 15 mM KCl, but genistein, also a broad range inhibitor of tyrosine kinases did inhibit the intracellular Ca(2+) concentration response to KCl and, concomitantly, decreased glutamate release evoked by the same stimulus, in a dose-dependent manner. These effects were not observed with the inactive analogue genistin. Therefore, we investigated the mechanism whereby genistein modulates Ca(2+) influx and glutamate release. Studies with voltage-gated Ca(2+) channel inhibitors showed that omega-conotoxin GVIA did not further inhibit glutamate release or the Ca(2+) influx stimulated by KCl in the presence of genistein. This tyrosine kinase inhibitor and omega-agatoxin IVA had a partially additive effect on those events. Nitrendipine did not reduce significantly the KCl-induced responses. Genistein further reduced Ca(2+) influx in response to KCl in the presence of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA, simultaneously. The effect of tyrosine phosphatase inhibitors was also tested on the influx of Ca(2+) and on glutamate release stimulated by KCl-depolarization. We found that the broad range inhibitors sodium orthovanadate and dephostatin did not significantly affect these KCl-evoked events.Our results suggest that genistein inhibits glutamate release and Ca(2+) influx in response to KCl independently of tyrosine kinase inhibition, and that tyrosine kinases and phosphatases are not key regulators of glutamate release in hippocampal nerve terminals.     

In vitro relaxation of rabbit and human internal anal sphincter by rutaecarpine, an alkaloid isolated from Evodia rutaecarpa

Rutaecarpine, a compound extracted from the Chinese medicinal herb Evodia rutaecarpa, has been shown to possess relaxing action on vascular smooth muscle from rat thoracic aorta. The internal anal sphincter is a specialized smooth muscle regulating important anorectal physiology. To investigate the effect and underlying mechanisms of rutaecarpine on internal anal sphincter, muscle strips from rabbit internal anal sphincter were used. The results showed that rutaecarpine (1 x 10(-10) M to 1 x 10(-4) M) produced a concentration-dependent muscular relaxation effect in our preparations, which were precontracted with acetylcholine. This muscular relaxation effect was not affected by treatment with L-N(G)-nitro-arginine methyl ester (a nitric oxide synthase inhibitor), methylene blue (a guanylate cyclase inhibitor), N-ethylmaleimide (an adenylate cyclase inhibitor), or by removal of the mucosa and submucosa tissue. Pretreatment with nifedipine (a calcium channel blocker) or extracellular Ca+2 removal by ethylenediaminetetraacetic acid (EDTA) greatly attenuated the relaxation effect, suggesting that calcium ion might be involved. In experiments using strips from human internal anal sphincter, an even more prominent relaxation effect was shown. It is thus concluded that rutaecarpine caused relaxation on internal anal sphincter from rabbits and human subjects. The relaxation action was not related to NO-cGMP pathway, instead calcium ion might play an important role and shed insight into clinical implications for those anorectal disorders with hyperactive anal tone.     

Genistein inhibits lysosomal enzyme release by suppressing Ca2+ influx in HL-60 granulocytes

The tyrosine kinase inhibitor genistein (5-200 microM) suppressed Ca(2+)-dependent fMLP (1 microM) and ATP (100 microM)-induced release of the lysosomal enzyme, beta-glucuronidase from neutrophil-like HL-60 granulocytes. Agonist-induced Ca2+ mobilization resulted from the release of intracellular Ca2+ stores and the influx of extracellular Ca2+. Genistein (200 microM) suppressed fMLP (1 microM) and ATP (100 microM)-induced Ca2+ mobilization, by 30-40%. Ca2+ release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+ (Mn2+) influx. Consistent with these findings, genistein (200 microM) or removal of extracellular Ca2+ (EGTA 1 mM), inhibited Ca(2+)-dependent agonist-induced beta-glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced beta-glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+ (Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+ influx induced by thapsigargin (0.5 microM). These data indicate that agonist-induced Ca2+ influx and store-operated Ca2+ influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL-60 granulocytes.     

Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder

We have evaluated the presence of capacitative Ca(2+) entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca(2+) channels. Changes in cytosolic Ca(2+) concentration induced by Ca(2+) entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca(2+) store pump, induced a transient Ca(2+) release followed by sustained entry of extracellular Ca(2+). Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca(2+)-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca(2+) resulted in a sustained increase in Ca(2+) entry on subsequent reapplication of Ca(2+). This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockade, pinacidil, and Gd(3+). Accumulation of the voltage-sensitive dye 3,3'-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockers, and Gd(3+). We conclude that, in GBSM, release of Ca(2+) from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca(2+) channels. This process may play a role in excitation-contraction coupling in GBSM.     

Contribution of store-operated Ca2+ entry to pHo-dependent changes in vascular tone of porcine coronary smooth muscle

Vascular smooth muscle contracts on increases of extracellular pH (pH(o)) and relaxes on pH(o) decreases possibly resulting from changes in transsarcolemmal Ca(2+) influx. Therefore, we studied store-operated Ca(2+) entry (SOCE; i.e. capacitative Ca(2+) entry (CCE)) during acidification (pH(o)=6.5) and alkalinization (pH(o)=8.0) in isolated porcine coronary smooth muscle cells (SMCs) by monitoring cytoplasmic Ca(2+) ([Ca(2+)](i)) and divalent cation entry (Mn(2+) quench) with fura-2/AM-fluorometry. Additionally, we evaluated the contribution of SOCE to pH(o)-dependent changes in isometric tension of porcine coronary smooth muscle strips. SOCE elicited in SMCs by the SERCA inhibitor BHQ was strongly modulated by pH(o) showing a decrease upon acidification and vice versa an increase upon alkalinization. BHQ-mediated tension of smooth muscle strips also revealed strong pH(o) dependence. In contrast, L-VOC-dependent tension ([K(+)](o)=20 and 40 mmol l(-1)) was remarkably less affected by pH(o) changes. Moreover, refilling of depleted Ca(2+) stores after repeated M(3)-cholinergic receptor stimulation could be almost completely inhibited by SKF 96365 and was markedly reduced by acidification and considerably enhanced by alkalinization pointing to a major role of SOCE in refilling. We conclude that vascular tone particularly responds to alterations in pH(o) whenever SOCE substantially contributes to the amount of activator Ca(2+) for contraction.