Guinea Pig Gallbladder Smooth Muscle Research Articles

Orai1 forms a signal complex with SK3 channel in gallbladder smooth muscle

Orai1 is one of the key components of store-operated Ca2+ entry (SOCE) involved in diverse physiological functions. Orai1 may associate with other proteins to form a signaling complex. In the present study, we investigated the interaction between Orai1 and small conductance Ca2+-activated potassium channel 3 (SK3). With the use of RNA interference technique, we found that the SOCE and its associated membrane hyperpolarization were reduced while Orai1 was knocked down by a specific Orai1 siRNA in guinea pig gallbladder smooth muscle. However, with the use of isometric tension measurements, our results revealed that agonist-induced muscle contractility was significantly enhanced after Orai1 protein was knocked down or the tissue was treated by SK3 inhibitor apamin, but not affected by larger conductance Ca2+-activated potassium channel inhibitor iberiotoxin or intermediate conductance Ca2+-activated potassium channel inhibitor TRAM-34. In addition, in the presence of apamin, Orai1 siRNA had no additional effect on agonist-induced contraction. In coimmunoprecipitation experiment, SK3 and Orai1 pulled down each other. These data suggest that, Orai1 physically associated with SK3 to form a signaling complex in gallbladder smooth muscle. Ca2+ entry via Orai1 activates SK3, resulting in membrane hyperpolarization in gallbladder smooth muscle. This hyperpolarizing effect of Orai1-SK3 coupling could serve to prevent excessive contraction of gallbladder smooth muscle in response to contractile agonists.

Sa1734 Relaxations Induced by Testosterone and Dihydrotestosterone in Isolated Guinea-Pig Gallbladder Smooth Muscle Strips

Sa1734 Relaxations Induced by Testosterone and Dihydrotestosterone in Isolated Guinea-Pig Gallbladder Smooth Muscle Strips

Mo1646 Mechanisms of Relaxant Activity by cGMP Stimuator in Guinea-Pig Gallbladder Smooth Muscle

The soluble guanylyl cyclase is expressed in guinea-pig gallbladder smooth muscle and agents that stimulate this enzyme activity cause gallbladder relaxation. The compound 5Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in gallbladder smooth muscle. This study investigated the mechanisms underlying the relaxations of guinea-pig gallbladder smooth muscle induced by BAY 41-2272. Gallbladder strips were mounted in 20-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed histamine-precontracted gallbladder strips. Prior incubation with the NO synthesis inhibitor L-NAME (0.1 mM) or the soluble guanylyl cyclase inhibitor ODQ (10 μM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. NO-donor, sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and almost completely inhibited by ODQ. To evaluate the effect of K+ channels, the relaxant responses of gallbladder muscle to Bay 41-2272 remained unchanged selective K+ channel inhibitor such as glibenclamide (10 μM), 4-aminopyridine (1 mM) or the co-incubation of charybdotoxin (0.1 μM) with apamin (1 μM). To evaluate the participation of prostanoids and cAMP pathway, the cyclooxygenase inhibitor indomethacin (10 μM) and selective phosphodiesterase type 4 inhibitor rolipram (1 μM) failed to significantly affect the BAY 41-2272-induced gallbladder relaxations. BAY 41-2272 shifted to the right the gallbladder contractile responses to either histamine (0.0001-10 μM). BAY 41-2272 (10 μM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2. The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 μM), or the PKG, Rp8-pCPT-cGMPS (10 μM) on relaxation induced by the cumulative adminstration of BAY 41-2272 were studied in precontracted with 1 μM histamine. The relaxing effect of BAY 41-2272 were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. These results indicate that BAY 41-2272 causes cGMP-dependent guinea-pig gallbladder smooth muscle relaxations in a synergistic fashion with NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca2+ entry blockade.

Mo1650 Purinergic Inhibitory Neuromuscular Transmission in Human Jejunum and Guinea Pig Colon

The soluble guanylyl cyclase is expressed in guinea-pig gallbladder smooth muscle and agents that stimulate this enzyme activity cause gallbladder relaxation. The compound 5Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in gallbladder smooth muscle. This study investigated the mechanisms underlying the relaxations of guinea-pig gallbladder smooth muscle induced by BAY 41-2272. Gallbladder strips were mounted in 20-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed histamine-precontracted gallbladder strips. Prior incubation with the NO synthesis inhibitor L-NAME (0.1 mM) or the soluble guanylyl cyclase inhibitor ODQ (10 μM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. NO-donor, sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and almost completely inhibited by ODQ. To evaluate the effect of K+ channels, the relaxant responses of gallbladder muscle to Bay 41-2272 remained unchanged selective K+ channel inhibitor such as glibenclamide (10 μM), 4-aminopyridine (1 mM) or the co-incubation of charybdotoxin (0.1 μM) with apamin (1 μM). To evaluate the participation of prostanoids and cAMP pathway, the cyclooxygenase inhibitor indomethacin (10 μM) and selective phosphodiesterase type 4 inhibitor rolipram (1 μM) failed to significantly affect the BAY 41-2272-induced gallbladder relaxations. BAY 41-2272 shifted to the right the gallbladder contractile responses to either histamine (0.0001-10 μM). BAY 41-2272 (10 μM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2. The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 μM), or the PKG, Rp8-pCPT-cGMPS (10 μM) on relaxation induced by the cumulative adminstration of BAY 41-2272 were studied in precontracted with 1 μM histamine. The relaxing effect of BAY 41-2272 were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. These results indicate that BAY 41-2272 causes cGMP-dependent guinea-pig gallbladder smooth muscle relaxations in a synergistic fashion with NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca2+ entry blockade.

Mo1649 Dopaminergic Control of Motility in Human and Guinea Pig Intestine

The soluble guanylyl cyclase is expressed in guinea-pig gallbladder smooth muscle and agents that stimulate this enzyme activity cause gallbladder relaxation. The compound 5Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in gallbladder smooth muscle. This study investigated the mechanisms underlying the relaxations of guinea-pig gallbladder smooth muscle induced by BAY 41-2272. Gallbladder strips were mounted in 20-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed histamine-precontracted gallbladder strips. Prior incubation with the NO synthesis inhibitor L-NAME (0.1 mM) or the soluble guanylyl cyclase inhibitor ODQ (10 μM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. NO-donor, sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and almost completely inhibited by ODQ. To evaluate the effect of K+ channels, the relaxant responses of gallbladder muscle to Bay 41-2272 remained unchanged selective K+ channel inhibitor such as glibenclamide (10 μM), 4-aminopyridine (1 mM) or the co-incubation of charybdotoxin (0.1 μM) with apamin (1 μM). To evaluate the participation of prostanoids and cAMP pathway, the cyclooxygenase inhibitor indomethacin (10 μM) and selective phosphodiesterase type 4 inhibitor rolipram (1 μM) failed to significantly affect the BAY 41-2272-induced gallbladder relaxations. BAY 41-2272 shifted to the right the gallbladder contractile responses to either histamine (0.0001-10 μM). BAY 41-2272 (10 μM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2. The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 μM), or the PKG, Rp8-pCPT-cGMPS (10 μM) on relaxation induced by the cumulative adminstration of BAY 41-2272 were studied in precontracted with 1 μM histamine. The relaxing effect of BAY 41-2272 were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. These results indicate that BAY 41-2272 causes cGMP-dependent guinea-pig gallbladder smooth muscle relaxations in a synergistic fashion with NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca2+ entry blockade.

Emodin Inhibits Voltage‐Dependent Potassium Current in Guinea Pig Gallbladder Smooth Muscle

Emodin is known to prompt bile secretion in gallbladder and to be used in the treatment of cholesterol stones. We studied the effects of emodin on the contraction of gallbladder smooth muscle and voltage-dependent K(+) current in gallbladder smooth muscle cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion, and K(+) current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of gallbladder smooth muscle strips and inhibited voltage-dependent K(+) current in a dose-dependent manner. When 10 microM emodin was applied to gallbladder smooth muscle cells for 3-6 min., the amplitude of voltage-dependent K(+) current was decreased by 31.5 +/- 0.5% at +40 mV, and this inhibitory effect mostly recovered after washout. The steady-state inactivation curves were shifted in a hyperpolarizing direction by emodin. In the presence of the protein kinase C inhibitors staurosporine and chelerythrine, the effect of emodin on voltage-dependent K(+ )current was significantly attenuated. In conclusion, emodin promotes gallbladder contraction, mainly by inhibiting voltage-dependent K(+) current via the protein kinase C pathway. These findings provide theoretical foundation for the application of emodin in gallbladder motility disorders.

Differences in Action of PACAP-27 and PACAP-38 on Guinea Pig Gallbladder Smooth Muscle Using Synthetic C-terminally Modified PACAP Peptides

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two bioactive forms, PACAP-38 and PACAP-27 that have identical N-terminal sequences but differ by the presence of a C-terminal 11 residue elongation in the former. Although VIP and PACAP have several similar biological actions due to their amino acid sequence similarity, we have found that they evoke opposite responses in the guinea pig gallbladder smooth muscle, where PACAP induces contraction while VIP causes relaxation. In addition the response to PACAP-38 is four times lower than that of PACAP-27. In a previous study we have reported the role of the N-terminal α-helical regions of PACAP-27 which play a key role in gallbladder contraction. In the present study the biological action on the guinea pig gallbladder was investigated using a synthetic mini-library of C-terminally deleted peptides related to PACAP-38. The effects caused by residues within the C-terminus are not a result of a response via the M-receptor or Na+ channel, but most likely arise from a delicate balance between the differential effects of PACAP-38 on specific PAC1 and VPACs receptors.

Developmental changes in Ca2+ homeostasis and contractility in gallbladder smooth muscle

Relatively little is known about the contribution of Ca(2+)-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied Ca(2+) homeostasis and Ca(2+) sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular Ca(2+) concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased Ca(2+) mobilization, suggesting immaturity of Ca(2+) sensitization mechanisms. The enhanced Ca(2+) release in the newborn gallbladder was the result of the increase in the size of the releasable Ca(2+) pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane Ca(2+) pump nor the Na(+)/Ca(2+) exchanger collaborate in the extrusion of Ca(2+). In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase pump. The reduced Ca(2+) sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation.

Emodin increases Ca 2+ influx through L-type Ca 2+ channel in guinea pig gallbladder smooth muscle

Emodin is known to be used in the treatment of cholesterol stones and cholecystitis. This study sought to investigate the effects of emodin on the contraction of gallbladder smooth muscle (GBSM), intracellular Ca 2+ concentration and L-type calcium current in GBSM cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion. Cells were loaded with fluo-3/AM and [Ca 2+] i was determined by a laser confocal microscope. Calcium current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of GBSM strips in a dose-dependent manner. Emodin elevated [Ca 2+] i in GBSM cells, and this effect was attenuated by pretreatment with nifedipine. In addition, Emodin increased L-type calcium current at concentrations of 1 to 30 µM (at + 10 mV, 10 µM, 45.1 ± 5.2% compared to control, EC 50 = 3.11 µM). In the presence of protein kinase C (PKC) inhibitor, Staurosporine, emodin did not significantly affect the calcium current. However, phorbol 12, 13-dibutyrate mimicked emodin in enhancement of the calcium current. These results suggest that emodin promotes gallbladder contraction by increasing Ca 2+ influx through L-type calcium channel via PKC pathway.

ATP induces guinea pig gallbladder smooth muscle excitability via the P2Y4 receptor and COX-1 activity

The purpose of this study was to elucidate the mechanisms by which ATP increases guinea pig gallbladder smooth muscle (GBSM) excitability. We evaluated changes in membrane potential and action potential (AP) frequency in GBSM by use of intracellular recording. Application of ATP (100 microM) caused membrane depolarization and a significant increase in AP frequency that were not sensitive to block by tetrodotoxin (0.5 microM). The nonselective P2 antagonist, suramin (100 microM), blocked the excitatory response, resulting in decreased AP frequency in the presence of ATP. The excitatory response to ATP was not altered by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (30 microM), a nonselective P2X antagonist. UTP also caused membrane depolarization and increased AP frequency, with a similar dose-response relationship as ATP. RT-PCR demonstrated that the P2Y(4), but not P2Y(2), receptor subtype is expressed in guinea pig gallbladder muscularis. ATP induced excitation was blocked by indomethacin (10 microM) and the cyclooxygenase (COX)-1 inhibitor SC-560 (300 nM), but not the COX-2 inhibitor nimesulide (500 nM). These data suggest that ATP stimulates P2Y(4) receptors within the gallbladder muscularis and, in turn, stimulate prostanoid production via COX-1 leading to increased excitability of GBSM.

Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle

Mitochondrial Ca(2+) handling has been implicated in spontaneous rhythmic activity in smooth muscle and interstitial cells of Cajal. In this investigation we evaluated the effect of mitochondrial inhibitors on spontaneous action potentials (APs), Ca(2+) flashes, and Ca(2+) waves in gallbladder smooth muscle (GBSM). Disruption of the mitochondrial membrane potential with carbonyl cyanide 3-chlorophenylhydrazone, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, rotenone, and antimycin A significantly reduced or eliminated APs, Ca(2+) flashes, and Ca(2+) waves in GBSM. Blockade of ATP production with oligomycin did not alter APs or Ca(2+) flashes but significantly reduced Ca(2+) wave frequency. Inhibition of mitochondrial Ca(2+) uptake and Ca(2+) release with Ru360 and CGP-37157, respectively, reduced the frequency of Ca(2+) flashes and Ca(2+) waves in GBSM. Similar to oligomycin, cyclosporin A did not alter AP and Ca(2+) flash frequency but significantly reduced Ca(2+) wave activity. These data suggest that mitochondrial Ca(2+) handling is necessary for the generation of spontaneous electrical activity and may therefore play an important role in gallbladder tone and motility.

Calcium controls smooth muscle TRPC gene transcription via the CaMK/calcineurin-dependent pathways

Transient receptor potential protein family C (TRPC) has been proposed as a candidate for channels involved in capacitative Ca(2+) entry (CCE) mechanisms, but the modulation of their gene expression remains unexplored. In this study we show that guinea pig gallbladder smooth muscle contains mRNA encoding TRPC1, TRPC2, TRPC3, and TRPC4 proteins whose abundance depends on cytosolic Ca(2+) level ([Ca(2+)](i)). Thus lowering the levels of cellular calcium with the chelators EGTA and BAPTA AM results in a downregulation of TRPC1-TRPC4 gene and protein expression. In contrast, activation of Ca(2+) influx through L-type Ca(2+) channels and Ca(2+) release from intracellular stores induced an increase in TRPC1-TRPC4 mRNA and protein abundance. Activation of Ca(2+)/calmodulin-dependent kinases (CaMK) and phosphorylation of cAMP-response element binding protein accounts for the increase in TRPC mRNA transcription in response to L-type channel-mediated Ca(2+) influx . In addition to this mechanism, activation of TRPC gene expression by intracellular Ca(2+) release also involves calcineurin pathway. According to the proposed role for these channels, activation of CCE induced an increase in TRPC1 and TRPC3 mRNA abundance, which depends on the integrity of the calcineurin and CaMK pathways. These findings show for the first time an essential autoregulatory role of Ca(2+) in Ca(2+) homeostasis at the level of TRPC gene and protein expression.

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and alpha-helical regions of PACAP-27 are required for initiating gallbladder contraction.

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Intracellular Ca(2+) waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca(2+) and voltage recordings with microelectrodes, respectively. Spontaneous Ca(2+) waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca(2+)-induced Ca(2+) release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca(2+) waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca(2+) waves were abolished by the Ins(1,4,5)P(3) receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca(2+) waves and induced Ca(2+) flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca(2+) waves depend on sarco(endo)plasmic reticulum (SR) Ca(2+) release via Ins(1,4,5)P(3) receptors, PLC activity, Ca(2+) influx via VDCCs, and SR Ca(2+) concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca(2+) release via Ins(1,4,5)P(3) receptors. Ca(2+) waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.

Role of Rho-kinase in guinea-pig gallbladder smooth muscle contraction

Guinea-pig gallbladder smooth muscle contractions can be elicited pharmacologically by a range of mechanisms. The involvement of Rho-kinase in contractions mediated by receptor-dependent and receptor-independent mechanisms was investigated using the Rho-kinase inhibitor (+)-( R)- trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexane carboxamide (Y-27632). In a separate series of experiments, the role of Rho-kinase in the contractile response to Ca 2+ entry through store-operated Ca 2+ channels and to electrical field stimulation was also examined. Y-27632 (10 μM), which caused a significant decrease ( P < 0.0005) in basal resting tone, significantly inhibited gallbladder contractions evoked by cumulative additions of the G-protein-coupled agonists, carbachol (1 nM–100 μM; P < 0.05) and cholecystokinin (10 nM–1 μM; P < 0.005). Y-27632 also inhibited the contractions evoked by a single addition of the sarcoplasmic reticulum ATPase inhibitor, thapsigargin (1 μM; P < 0.0005) and cumulative additions of KCl (10–85 mM; P < 0.0005). The contractile response to Ca 2+ entry through store-operated Ca 2+ channels was significantly inhibited by Y-27632 ( P < 0.05) as were the contractile responses evoked by electrical field stimulation (2–25 Hz; P < 0.0005). In contrast, Y-27632 had no significant effect on contractions evoked by phorbol 12,13-dibutyrate (0.1 nM–1 μM; a protein kinase C activator) or by the phosphatase inhibitor, cantharidin (100 μM). In conclusion, Rho-kinase contributes to the contractile response in guinea-pig gallbladder smooth muscle evoked by both G-protein-coupled and non-G-protein-coupled mechanisms in addition to contributing to the maintenance of basal tone. It also contributes to the contractile responses resulting from electrical field stimulation and store-operated Ca 2+ channel entry.

Spontaneous electrical rhythmicity and the role of the sarcoplasmic reticulum in the excitability of guinea pig gallbladder smooth muscle cells

Spontaneous action potentials and Ca(2+) transients were investigated in intact gallbladder preparations to determine how electrical events propagate and the cellular mechanisms that modulate these events. Rhythmic phasic contractions were preceded by Ca(2+) flashes that were either focal (limited to one or a few bundles), multifocal (occurring asynchronously in several bundles), or global (simultaneous flashes throughout the field). Ca(2+) flashes and action potentials were abolished by inhibiting sarcoplasmic reticulum (SR) Ca(2+) release via inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] channels with 2-aminoethoxydiphenyl borate and xestospongin C or by inhibiting voltage-dependent Ca(2+) channels (VDCCs) with nifedipine or diltiazem or nisoldipine. Inhibiting ryanodine channels with ryanodine caused multiple spikes superimposed upon plateaus of action potentials and extended quiescent periods. Depletion of SR Ca(2+) stores with thapsigargin or cyclopiazonic acid increased the frequency and duration of Ca(2+) flashes and action potentials. Acetylcholine, carbachol, or cholecystokinin increased synchronized and increased the frequency of Ca(2+) flashes and action potentials. The phospholipase C (PLC) inhibitor U-73122 did not affect Ca(2+) flash or action potential activity but inhibited the excitatory effects of acetylcholine on these events. These results indicate that Ca(2+) flashes correspond to action potentials and that rhythmic excitation in the gallbladder is multifocal among gallbladder smooth muscle bundles and can be synchronized by excitatory agonists. These events do not depend on PLC activation, but agonist stimulation involves activation of PLC. Generation of these events depends on Ca(2+) entry via VDCCs and on Ca(2+) mobilization from the SR via Ins(1,4,5)P(3) channels.

Changes in guinea pig gallbladder smooth muscle Ca2+homeostasis by acute acalculous cholecystitis

Impaired smooth muscle contractility is a hallmark of acute acalculous cholecystitis. Although free cytosolic Ca2+ ([Ca2+]i) is a critical step in smooth muscle contraction, possible alterations in Ca2+ homeostasis by cholecystitis have not been elucidated. Our aim was to elucidate changes in the Ca2+ signaling pathways induced by this gallbladder dysfunction. [Ca2+]i was determined by epifluorescence microscopy in fura 2-loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. F-actin content was quantified by confocal microscopy. Ca2+ responses to the inositol trisphosphate (InsP3) mobilizing agonist CCK and to caffeine, an activator of the ryanodine receptors, were impaired in cholecystitic cells. This impairment was not the result of a decrease in the size of the releasable pool. Inflammation also inhibited Ca2+ influx through L-type Ca2+ channels and capacitative Ca2+ entry induced by depletion of intracellular Ca2+ pools. In addition, the pharmacological phenotype of these channels was altered in cholecystitic cells. Inflammation impaired contractility further than Ca2+ signal attenuation, which could be related to the decrease in F-actin that was detected in cholecystitic smooth muscle cells. These findings indicate that cholecystitis decreases both Ca2+ release and Ca2+ influx in gallbladder smooth muscle, but a loss in the sensitivity of the contractile machinery to Ca2+ may also be responsible for the impairment in gallbladder contractility.

Cholecystokinin (CCK) down regulates PGE 2 and PGI 2 release in inflamed Guinea pig gallbladder smooth muscle cell cultures

This study examines the hypothesis that cholecystitis down-regulates Guinea pig gallbladder (GPGB) smooth muscle cholecystokinin (CCK)-stimulated prostaglandin (PG) release. Guinea pig gallbladder from Control and 48 h bile duct ligated (BDL) animals were placed in cell culture and grown to confluence. The cultures underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacyclin Synthase (PS), or were incubated with CCK at 10 −8 M or 10 −6 M with and without indomethacin for 1 h and analyzed for release of 6-keto-PGF 1 α , PGE 2 and TxB 2 by EIA. BDL increased Guinea pig gallbladder cell culture basal PGE 2 and PGI 2 release which was in part due to increased COX-2 content. CCK incubation down-regulated BDL Guinea pig gallbladder cell culture release of 6-keto-PGF 1 α and PGE 2 and down-regulated COX-2 content but did not alter the Control group. The decrease in CCK-mediated BDL cell Guinea pig gallbladder release may be an endogenous mechanism to limit physiologic derangements induced by increased endogenous gallbladder PG synthesis during early acute cholecystitis.

Enhancement of EFS-induced contractions, by agmatine, in guinea pig gallbladder smooth muscle strips

Electrical field stimulation of gallbladder muscle strips produces frequency-dependent contractions by activating cholinergic nerves. The cholinergic motor function of the gallbladder and enteric system is also modulated by other mediators. The aim of this study was to investigate the role of agmatine, a ligand for alpha 2-adrenoceptors and imidazoline binding sites, in the cholinergic motor activity of guinea pig gallbladder smooth muscle. Gallbladder muscle strips obtained from guinea pigs were subjected to electrical field stimulation (1-64 Hz, 100 V, 1-ms pulse width, and 10-s train duration). Frequency-response contractions of gallbladder muscle strips were traced before and after the addition of cumulative concentrations of agmatine (10(-5)-10(-3) M) to the tissue bath. The same set of experiments was repeated in the presence of different antagonists. Agmatine by itself did not produce any contractions in guinea pig gallbladder muscle strips, but significantly enhanced the contractile response produced by electrical field stimulation. Yohimbine (10(-6) M), a selective alpha 2-adrenergic blocker, neither decreased nor increased the enhancement induced by agmatine. However, idazoxan (10(-4) M), an alpha-receptor blocker and imidazoline receptor antagonist, abolished this enhanced contractile response. Pretreatment with N(W)-nitro L-arginine methyl ester (L-NAME; 30 microM), and indomethacin (10 microM) did not inhibit the effect of agmatine. Our findings indicate that agmatine has a modulator role in the electrical field stimulation-induced cholinergic contractions of guinea pig gallbladder smooth muscle strips, and this role could be mediated by imidazoline receptors. Receptor binding studies should be done to determine the presence of endogenous agmatine and imidazoline receptors in gallbladder smooth muscle.

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.