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Highlighting the importance of sex as a biological variable, we recently reported sex differences in guinea pig in vivo electrocardiogram (ECG) measurements. However, substantial inconsistencies exist in this animal model, with conflicting reports of sex-specific differences in cardiac electrophysiology observed in vivo and in vitro. Herein, we evaluated whether sexual dimorphism persists in ex vivo preparations, using an isolated intact heart preparation. Pseudo-ECG recordings were collected in conjunction with dual optical mapping of transmembrane voltage and intracellular calcium from Langendorff-perfused hearts. In contrast to our in vivo results, we did not observe sex-specific differences in ECG parameters collected from isolated hearts. Furthermore, we observed significant age-specific differences in action potential duration (APD) and Ca 2+ transient duration (CaD) during both normal sinus rhythm (NSR) and in response to dynamic pacing but only a modest sex-specific difference in CaD 30 . Similarly, the alternans fluctuation coefficient, conduction velocity during sinus rhythm or in response to pacing, and electrophysiology parameters (atrioventricular nodal effective refractory period, Wenckebach cycle length) were comparable between males and females. Results of our study suggest that the observed sex-specific differences in in vivo ECG parameters from guinea pigs are diminished in ex vivo isolated heart preparations, although age-specific patterns are prevalent. To assess sex as a biological variable in cardiac electrophysiology, a comprehensive approach may be necessary using both in vitro measurements from cardiomyocyte or intact heart preparations with secondary follow-up in vivo studies. NEW & NOTEWORTHY We evaluated whether the guinea pig heart has intrinsic sex-specific differences in cardiac electrophysiology. Although we observed sex-specific differences in in vivo ECGs, these differences did not persist ex vivo. Using a whole heart model, we observed similar APD, CaD, conduction velocity, and alternans susceptibility in males and females. We conclude that sex-specific differences in guinea pig cardiac electrophysiology are likely influenced by the in vivo environment and less dependent on the intrinsic electrical properties of the heart.

Colonic motor complexes (CMCs) are a major neurogenic activity in guineapig distal colon. The identity of the enteric neurons that initiate this activity is not established. Specialized intrinsic primary afferent neurons (IPANs) are a major candidate. We aimed to test this hypothesis. To do this, segments of guineapig distal colon were suspended vertically in heated organ baths and propulsive forces acting on a pellet inside the lumen were recorded by isometric force transducer while pharmacological agents were applied to affect IPAN function. In the absence of drugs, CMCs acted periodically on the pellet, generating peak propulsive forces of 12.7 ± 5 g at 0.56 ± 0.22 cpm, lasting 49 ± 17 s (215 preparations; n = 60). Most but not all CMCs were abolished by nicotinic receptor blockade to inhibit fast excitatory synaptic transmission (50/62 preparations; n = 25). Remarkably, CMCs inhibited by hexamethonium were restored by a pharmacological strategy that aimed to enhance IPAN excitability. Thus, CMCs were restored by increased smooth muscle tension (using BAY K8644, bethanechol or carbachol) and by IPAN excitation using phorbol dibutyrate; NK3 receptor agonist, senktide; and partially by αCGRP. The IPAN inhibitor, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (DCEBIO), decreased CMC frequency. CGRP, but not NK3-receptor antagonists, decreased CMC frequency in naive preparations. Finally, CMCs were blocked by tetrodotoxin, and this was not reversed by any drugs listed above. These results support a major role for IPANs that does not require fast synaptic transmission, in the periodic initiation of neurogenic propulsive contractions. Endogenous CGRP plays a role in determining CMC frequency, whereas further unidentified signaling pathways may determine their amplitude and duration. NEW & NOTEWORTHY The colonic motor complex (CMC) initiates propulsion in guinea pig colon. Here, CMCs evoked by an intraluminal pellet were restored during nicotinic receptor blockade by pharmacological agents that directly or indirectly enhance intrinsic primary afferent neuron (IPAN) excitability. IPANs are the only enteric neuron in colon that contain CGRP. Blocking CGRP receptors decreased CMC frequency, implicating their role in CMC initiation. The results support a role for IPANs in the initiation of CMCs.

Bile acid reflux in the esophagus plays a role in the pathogenesis of certain esophageal disorders, where it can induce esophageal pain and heartburn. The present study aimed to determine whether bile acid, deoxycholic acid (DCA), directly activates and sensitizes esophageal vagal nociceptive afferent C-fiber subtypes. DCA-elicited effects on vagal nodose and jugular neurons were studied by calcium imaging. Its effects on esophageal-labeled nodose and jugular neurons were then determined by patch-clamp recording. At nodose and jugular C-fiber nerve endings in the esophagus, DCA-evoked action potentials (APs) were compared by extracellular single-unit recordings in ex vivo esophageal-vagal preparations. DCA application induced calcium influxes in nodose and jugular neurons and elicited inward currents in esophageal-labeled nodose and jugular neurons. In the presence of DCA, the current densities elicited by capsaicin were enhanced in those labeled neurons. Consistently, DCA perfusion at nerve terminals in the esophagus evoked APs in about 50% of esophageal nodose and jugular C-fibers. In DCA-sensitive C-fibers, DCA perfusion also sensitized the fibers such that the subsequent response to capsaicin was amplified. Collectively, these results provide new evidence that DCA directly activates and sensitizes nociceptive nodose and jugular C-fibers in the esophagus. Such activation and sensitization effects may contribute to bile acid-induced esophageal nociceptive symptoms that are refractory to proton-pump inhibitor therapy. NEW & NOTEWORTHY Bile acid reflux in the esophagus can induce pain and heartburn in certain esophageal disorders, but the underlying neuronal mechanism is still unclear. The present study demonstrated that bile acid, deoxycholic acid (DCA), directly activates esophageal vagal afferent nodose and jugular nociceptive C-fibers and sensitizes their response to capsaicin. Such effects may contribute to bile acid-induced esophageal nociceptive symptoms that refractory to proton-pump inhibitors (PPIs) therapy.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases. NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Bile acid reflux in the esophagus plays a role in the pathogenesis of certain esophageal disorders, where it can induce esophageal pain and heartburn. The present study aimed to determine whether bile acid, deoxycholic acid (DCA), directly activates and sensitizes esophageal vagal nociceptive afferent C-fiber subtypes. DCA-elicited effects on vagal nodose and jugular neurons were studied by calcium imaging. Its effects on esophageal-labeled nodose and jugular neurons were then determined by patch-clamp recording. At nodose and jugular C-fiber nerve endings in the esophagus, DCA-evoked action potentials (APs) were compared by extracellular single-unit recordings in ex vivo esophageal-vagal preparations. DCA application induced calcium influxes in nodose and jugular neurons and elicited inward currents in esophageal-labeled nodose and jugular neurons. In the presence of DCA, the current densities elicited by capsaicin were enhanced in those labeled neurons. Consistently, DCA perfusion at nerve terminals in the esophagus evoked APs in about 50% of esophageal nodose and jugular C-fibers. In DCA-sensitive C-fibers, DCA perfusion also sensitized the fibers such that the subsequent response to capsaicin was amplified. Collectively, these results provide new evidence that DCA directly activates and sensitizes nociceptive nodose and jugular C-fibers in the esophagus. Such activation and sensitization effects may contribute to bile acid-induced esophageal nociceptive symptoms that are refractory to proton-pump inhibitor therapy. [ABSTRACT FROM AUTHOR]

Nonselective cation channels, consistent with transient receptor potential melastatin-4 (TRPM4), regulate detrusor smooth muscle (DSM) function. TRPM4 channels can exist as homomers or assemble with sulfonylurea receptors (SURs) as complexes. We evaluated contributions of TRPM4/SUR-TRPM4 channels to DSM excitability and contractility by examining the effects of TRPM4/SUR-TRPM4 channel modulators 9-phenanthrol, glibenclamide, and diazoxide on freshly-isolated guinea pig DSM cells (amphotericin-B perforated patch-clamp electrophysiology) and mucosa-free DSM strips (isometric tension recordings). In DSM cells, complete removal of extracellular Na + decreased voltage-step-induced cation (non-K + selective) currents. At high positive membrane potentials, 9-phenanthrol at 100 μM attenuated voltage step-induced currents more effectively than at 30 μM, revealing concentration-dependent, voltage-sensitive inhibition. In comparison to 9-phenanthrol, glibenclamide (100 μM) displayed lower inhibition of cation currents. In the presence of glibenclamide (100 μM), 9-phenanthrol (100 μM) further decreased the currents. The SUR-TRPM4 complex activator diazoxide (100-300 μM) weakly inhibited the currents. 9-Phenanthrol, but not glibenclamide or diazoxide, increased cell capacitance (a cell surface area indicator). In contractility studies, glibenclamide displayed lower potencies than 9-phenanthrol attenuating spontaneous and 20 mM KCl-induced DSM phasic contractions. While both compounds showed similar maximum inhibitions on DSM spontaneous phasic contractions, glibenclamide was generally less efficacious on 20 mM KCl-induced phasic contractions. In summary, the observed differential effects of 9-phenanthrol and glibenclamide on DSM excitability and contractility support unique mechanisms for the two compounds. The data suggest that SUR-TRPM4 complexes do not contribute to DSM function. This study advances our understanding of pharmacological effects of glibenclamide and 9-phenanthrol on DSM cell cation currents.

Levosimendan is a calcium sensitizer developed for the treatment of heart failure. It increases contractile force by enhancing the sensitivity of myofilaments to calcium. Besides this sensitizing effect, the drug has also been reported to show some inhibitory action on phosphodiesterase 3 (PDE3). The inotropic effects of levosimendan have been studied on guinea pig paced perfused hearts by using modular control analysis (MoCA) (Diolez P, Deschodt-Arsac V, Raffard G, Simon C, Santos PD, Thiandiere E, Arsac L, Franconi JM. Am J Physiol Regul Integr Comp Physiol 293: R13-R19, 2007.), an integrative approach of heart energetics using noninvasive [sup.31]p NMR. The aim was to evaluate quantitatively the respective effects of this drug on energy supply and demand modules. Under our experimental conditions, 0.7 [micro]M levosimendan induced a 45% increase in paced heart output associated with a 7% decrease in phosphocreatine and a negligible increase in oxygen consumption. Because MoCA allows in situ study of the internal regulations in intact beating heart energetics, it was applied to describe quantitatively by which routes levosimendan exerts its inotropic action. MoCA demonstrated the absence of any significant effect of the drug on the supply module, which is responsible for the lower increase in oxygen consumption, compared with epinephrine, which increases the ratio between myocardial oxygen consumption and cardiac contraction. This result evidences that, under our conditions, a possible effect of levosimendan on PDE3 activity and/or intracellular calcium remains very low on mitochondrial activity and insignificant on integrated cardiac energetics. Thus, levosimendan inotropic effect on guinea pig heart depends almost entirely on the calcium[-sensitizing properties leading to myofilament activation and the concomitant activation of energy supply by the decrease in PCr, therefore improving energetic efficiency of contraction. systems biology; modular control analysis; calcium sensitizer; mitochondria; phosphate-31 magnetic resonance spectroscopy; oxygen consumption doi: 10.1152/ajpregu.00184.2010.

To explore the precise mechanisms of the inhibitory effects of blebbistatin, a potent inhibitor of myosin II, on smooth muscle contraction, we studied the blebbistatin effects on the mechanical properties and the structure of contractile filaments of skinned (cell membrane permeabilized) preparations from guinea pig taenia cecum. Blebbistatin at 10 [micro]M or higher suppressed [Ca.sup.2+]-induced tension development at any given [Ca.sup.2+] concentration but had little effects on the [Ca.sup.2+]-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.75 mM [Mg.sup.2+]-induced, 'myosin light chain phosphorylation-independent' tension development at more than 10 [micro]M. Furthermore, blebbistatin induced conformational change of smooth muscle myosin (SMM) and disrupted arrangement of SMM and thin filaments, resulting in inhibition of actin-SMM interaction irrespective of activation with [Ca.sup.2+]. In addition, blebbistatin partially inhibited [Mg.sup.2+]-ATPase activity of native actomyosin from guinea pig taenia cecum at around 10 [micro]M. These results suggested that blebbistatin suppressed skinned smooth muscle contraction through disruption of structure of SMM by the agent. smooth muscle; skinned preparations; contractile filaments; adenosinetriphosphatase doi: 10.1152/ajpcell.00269.2009.

Glucagon-like peptide-2 (GLP-2) is an important neuroendocrine peptide in intestinal physiology. It influences digestion, absorption, epithelial growth, motility, and blood flow. We studied involvement of GLP-2 in intestinal mucosal secretory behavior. Submucosal-mucosal preparations from guinea pig ileum were mounted in Ussing chambers for measurement of short-circuit current ([I.sub.sc]) as a surrogate for chloride secretion. GLP-2 action on neuronal release of acetylcholine was determined with ELISA. Enteric neuronal expression of the GLP-2 receptor (GLP-2R) was studied with immunohistochemical methods. Application of GLP-2 (0.1-100 nM) to the serosal or mucosal side of the preparations evoked no change in baseline [I.sub.sc] and did not alter transepithelial ionic conductance. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in [I.sub.sc], with an initially rapid rising phase followed by a sustained phase. Application of GLP-2 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-2R antagonist GLP-2-(3-33) significantly reversed suppression of the EFS-evoked responses by GLP-2. Tetrodotoxin, scopolamine, and hexamethonium, but not vasoactive intestinal peptide type 1 receptor (VPAC1) antagonist abolished or reduced to near zero the EFS-evoked responses. GLP-2 suppressed EFS-evoked acetylcholine release as measured by ELISA. Pretreatment with GLP-2(3-33) offset this action of GLP-2. In the submucosal plexus, GLP-2R immunoreactivity (-IR) was expressed in choline acetyltransferase-IR neurons, somatostatin-IR neurons, neuropeptide Y-IR neurons, and vasoactive intestinal peptide-IR neurons. We conclude that submucosal neurons in the guinea pig ileum express GLP-2R Activation of GLP-2R decreases neuronally evoked epithelial chloride secretion by suppressing acetylcholine release from secretomotor neurons. enteric nervous system; gastrointestinal hormones; intestine; mucosal secretion doi: 10.1152/ajpgi.00170.2009

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated by PACAP-selective [PAC.sub.1] receptors. In dissociated guinea pig cardiac neurons, PACAP causes a positive shift of the voltage dependence of activation of the hyperpolarization-activated nonselective cation current ([I.sub.h]). This observation suggested that an enhancement of [I.sub.h] contributed to the increase in excitability in neurons within whole-mount cardiac ganglia preparations. To evaluate the role of [I.sub.h] in the PACAP-induced increase in excitability, we compared the increase in action potentials generated by 10 nM PACAP in control neurons and in neurons treated with ZD7288 (10 or 100 [micro]M) or CsCl (2 or 2.5 mM), drugs known to inhibit [I.sub.h]. In control cells exposed to PACAP, I-s depolarizing current pulses elicited multiple action potential firing in 79% of the neurons. In ZD7288- or CsCl-containing solutions, the 10 nM PACAP-induced increase in excitability was markedly suppressed, with 7% and 21% of the neurons generating multiple action potentials, respectively. Prior results indicated that PACAP initiates depolarization by activating an inward current, which is separate from its enhancement of [I.sub.h]. Here, we show that a PACAP-induced depolarization was comparable in control neurons and neurons bathed in a CsCl-containing solution, an observation indicating that CsCl did not interfere with activation of the [PAC.sub.1] receptor by PACAP. Additional experiments indicated that pretreatment with the putative M current ([I.sub.M]) inhibitor 1 mM Ba[Cl.sub.2], but not 10 [micro]M XE991, initiated multiple firing in a majority of neurons, with resting potentials maintained at approximately -60 mV. Furthermore, in [Ba.sub.2+]-treated cells, 10 nM PACAP increased the number of action potentials generated. Our results indicate that PACAP enhancement of [I.sub.h], rather than inhibition of [I.sub.M] and other 1 mM [Ba.sub.2+]-sensitive [K.sub.+] currents, is a key ionic mechanism contributing to the peptide-induced increase in excitability for neurons within wholemount cardiac ganglia preparations. hyperpolarization-activated nonselective cation current; neuronal excitability; neuropeptide; parasympathetic neurons

Sensitization of esophageal sensory afferents by inflammatory mediators plays an important role in esophageal nociception. We have shown esophageal mast cell activation induces long-lasting mechanical hypersensitivity in vagal nodose C-fibers. However, the roles of mast cell mediators and downstream ion channels in this process are unclear. Mast cell tryptase via protease-activated receptor 2 (PAR2)-mediated pathways sensitizes sensory nerves and induces hyperalgesia. Transient receptor potential A1 (TRPA1) plays an important role in mechanosensory transduction and nociception. Here we tested the hypothesis that mast cell activation via a PAR2-dependent mechanism sensitizes TRPA1 to induce mechanical hypersensitivity in esophageal vagal C-fibers. The expression profiles of PAR2 and TRPA1 in vagal nodose ganglia were determined by immunostaining, Western blot, and RT-PCR. Extracellular recordings from esophageal nodose neurons were performed in ex vivo guinea pig esophageal-vagal preparations. Action potentials evoked by esophageal distention and chemical perfusion were compared. Both PAR2 and TRPA1 expressions were identified in vagal nodose neurons by immunostaining, Western blot, and RT-PCR. Ninety-one percent of TRPA1-positive neurons were of small and medium diameters, and 80% coexpressed PAR2. Esophageal mast cell activation significantly enhanced the response of nodose C-fibers to esophageal distension (mechanical hypersensitivity). This was mimicked by PAR2-activating peptide, which sustained for 90 min after wash, but not by PAR2 reverse peptide. TRPA1 inhibitor HC-030031 pretreatment significantly inhibited mechanical hypersensitivity induced by either mast cell activation or PAR2 agonist. Collectively, our data provide new evidence that sensitizing TRPA1 via a PAR2-dependent mechanism plays an important role in mast cell activation-induced mechanical hypersensitivity of vagal nodose C-fibers in guinea pig esophagus. visceral afferent; sensitization; nociception; protease-activated receptor 2; transient receptor potential A1

Pancreatic ductal epithelium produces a HC[O.sup.-.sub.3]-rich fluid. HC[O.sup.-.sub.3] transport across ductal apical membranes has been proposed to be mediated by both SLC26-mediated [Cl.sup.-]/HC[O.sup.-.sub.3] exchange and CFTR-mediated HC[O.sup.-.sub.3] conductance, with proportional contributions determined in part by axial changes in gene expression and luminal anion composition. In this study we investigated the characteristics of apical [Cl.sup.-]/ HCO3 exchange and its functional interaction with Cftr activity in isolated interlobular ducts of guinea pig pancreas. BCECF-loaded epithelial cells of luminally microperfused ducts were alkalinized by acetate prepulse or by luminal [C1.sup.-] removal in the presence of HC[O.sup.-.sub.3]-C[O.sub.2]. Intracellular pH recovery upon luminal [Cl.sup.-] restoration (nominal [Cl.sup.-]/HC[O.sup.-.sub.3] exchange) in cAMP-stimulated ducts was largely inhibited by luminal dihydro-DIDS ([H.sub.2]DIDS), accelerated by luminal CFTR inhibitor inh-172 (CFTRinh-172), and was insensitive to elevated bath [K.sup.+] concentration. Luminal introduction of CP'TRinh-172 into sealed duct lumens containing BCECF-dextran in HC[O.sup.-.sub.3]-free, [Cl.sup.-]-rich solution enhanced cAMP-stimulated HC[O.sup.-.sub.3] secretion, as calculated from changes in luminal pH and volume. Luminal [Cl.sup.-] removal produced, after a transient small depolarization, sustained cell hyperpolarization of ~15 mV consistent with electrogenic [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. The hyperpolarization was inhibited by [H.sub.2]DIDS and potentiated by CPTRinh-172. Inteflobular ducts expressed mRNAs encoding CPTR, Slc26a6, and Slc26a3, as detected by RT-PCR. Thus [Cl.sup.-]-dependent apical HC[O.sup.-sub.3] secretion in pancreatic duct is mediated predominantly by an Slc26a6-1ike [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger and is accelerated by inhibition of CFTR. This study demonstrates functional coupling between Cftr and Slc26a6-1ike [Cl.sup.-]/HC[O.sup.-.sub.3] exchange activity in apical membrane of guinea pig pancreatic interlobular duct. bicarbonate; Slc26; cystic fibrosis transmembrane conductance regulator; forskolin; [H.sub.2]DIDS

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 [micro]M) caused membrane depolarization and a significant increase in AP frequency that were not sensitive to block by tetrodotoxin (0.5 [micro]M). The nonselective P2 antagonist, suramin (100 [micro]M), 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 [micro]M), 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 P2[Y.sub.4], but not P2[Y.sub.2], receptor subtype is expressed in guinea pig gallbladder muscularis. ATP induced excitation was blocked by indomethacin (10 [micro]M) 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 P2[Y.sub.4] receptors within the gallbladder muscularis and, in turn, stimulate prostanoid production via COX-1 leading to increased excitability of GBSM. biliary motility; cyclooxygenase; purinergic; P2Y; prostaglandins

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-I antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane [B.sub.2] was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age. airway hyperresponsiveness; bronchospasm removal; cyclooxygenase; lipoxygenase; ontogenesis

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detmsor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladde,' detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the [Ca.sup.2+] indicator fluo 4AM. ICC fired [Ca.sup.2+] transients in response to stimulation by carbachol (1/10 [micro]M). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 [micro]M), an [M.sub.3] receptor antagonist, but not by the [M.sub.2] receptor antagonist methoctramine (1 [micro]M). The source of [Ca.sub.2+] underlying the carbachol transients in isolated 1CC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 [micro]M) or [Ni.sup.2+] (30-100 [micro]M) to block [Ca.sup.2+] channels or the removal of external [Ca.sup.2+] reduced the amplitude of the carbachol transients. Application of ryanodine (30 [micro]M) or tetracaine (100 [micro]M) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 [micro]M), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 [micro]M) caused a significant reduction and Xestospongin C (1 [micro]M) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a [Ca.sup.2+] indicator showed distinctively different patterns of spontaneous [Ca.sup.2+] events in smooth muscle ceils and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing [Ca.sup.2+] transients. confocal microscopy; c-Kit

Although [[beta].sub.2]-adrenoceptors represent 15-25% of [beta]-adrenoceptors in the guinea pig heart, their functionality is controversial. We assessed the inotropic effects of [[beta].sub.2]-adrenoceptor partial agonists in right papillary muscles. Salbutamol induced a small but significant concentration-dependent negative inotropic effect (NIE, -5% at 60 nM) followed by a moderate positive inotropic effect (+ 36% at 6 [micro]M) due to activation of [[beta].sub.1]-adrenoceptors. In the presence of 4 [micro]M atenolol, the concentration-dependent NIE (- 12% at 6 [micro]M) was biphasic, best described by a double logistic equation with respective [EC.sub.50] values of 3 and ~420 nM, and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI-118551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a prerequisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/phosphatidylinositol 3-kinase/ cytosolic phospholipase [A.sub.2] ([cPLA.sub.2]) activations leading to nitric oxide (NO) release via the arachidonic acid/cyclooxygenase pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at submicromolar concentrations, which is negatively controlled by [cPLA.sub.2]. The possibility that these effects are due to an equilibrium between different affinity states of the receptor ([G.sub.s]/[G.sub.i] coupled and [G.sub.i] independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that [[beta].sub.2]-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state. salbutamol; [beta]-adrenoceptor antagonists; cardiac contractility; [G.sub.s]/ -[G.sub.i] coupling; active conformations

The tonic contraction of human and guinea pig gallbladder (GB) is dependent on basal levels of [PGE.sub.2] and thromboxane [A.sub.2] (Tx[A.sub.2]). The pathway involved in the genesis of these prostaglandins has not been elucidated. We aimed to examine the source of reactive oxygen species (ROS) and whether they contribute to the genesis of GB tonic contraction by generating basal prostaglandin levels. Tonic contraction was studied in human and guinea pig GB muscle strips treated with ROS scavengers (Tiron and catalase), apocynin (an inhibitor of NADPH oxidase), and NOX-1 small interference RNA (siRNA). The subunits of NADPH oxidase and their functional roles were determined with specific antibodies in GB muscle cells. ROS scavengers reduced the GB tonic contraction and [H.sub.2][O.sub.2] and [PGE.sub.2] levels. Apocynin also inhibited the tonic contraction. Antibodies against subunits of NADPH oxidase present in GB muscle cells lowered [H.sub.2][O.sub.2] and [PGE.sub.2] levels. NOX-1 siRNA transfection reduced the tonic contraction, NOX-1 expression, and levels of [H.sub.2][O.sub.2] and [PGE.sub.2]. Tiron and apocynin inhibited the expected increase in tension and [H.sub.2][O.sub.2] levels induced by stretching of muscle strips. [H.sub.2][O.sub.2] increased the levels of [PGE.sub.2] and Tx[A.sub.2] by increasing platelet-activating factor-like lipids that phosphorylate p38 and [cPLA.sub.2] sequentially. [H.sub.2][O.sub.2] generated by NADPH oxidase participates in a signal transduction pathway that maintains the GB tonic contraction by activating PAF, p38, and [cPLA.sub.2] to generate prostaglandins. muscle; NADPH oxidase; prostaglandins

It was previously demonstrated that transmural electrophysiological heterogeneities can inscribe the ECG T wave. However, the bifurcated T wave caused by loss of inward rectifier potassium current ([I.sub.K1]) function is not fully explained by transmural heterogeneities. Since right ventricular (RV) guinea pig myocytes have significantly lower [I.sub.K1] than left ventricular (LV) myocytes, we hypothesized that the complex ECG can be inscribed by heterogeneous chamber-specific responses to hypokalemia and partial [I.sub.K1] blockade. Ratiometric optical action potentials were recorded from the epicardial surface of the RV and LV. Ba[Cl.sub.2] (10 [micro]mol/l) was perfused to partially block [I.sub.K1] in isolated guinea pig whole heart preparations. Ba[Cl.sub.2] or hypokalemia alone significantly increased RV basal (R[V.sub.B]) action potential duration (APD) by ~30% above control compared with LV apical (L[V.sub.A]) APD (14%, P < 0.05). In the presence of Ba[Cl.sub.2], 2 mmol/l extracellular potassium (hypokalemia) further increased R[V.sub.B] APD to a greater extent (31%) than L[V.sub.A] APD (19%, P < 0.05) compared with Ba[Cl.sub.2] perfusion alone. Maximal dispersion between R[V.sub.B] and L[V.sub.A] APD increased by 105% (P < 0.05), and the QT interval prolonged by 55% (P < 0.05) during hypokalemia and Ba[Cl.sub.2]. Hypokalemia and Ba[Cl.sub.2] produced an ECG with a double repolarization wave. The first wave (QT1) corresponded to selective depression of apical LV plateau potentials, while the second wave (QT2) corresponded to the latest repolarizing RVB myocytes. These data suggest that final repolarization is more sensitive to extracellular potassium changes in regions with reduced [I.sub.K1], particularly when [I.sub.K1] availability is reduced. Furthermore, underlying [I.sub.K1] heterogeneities can potentially contribute to the complex ECG during [I.sub.K1] loss of function and hypokalemia. electrophysiology; waves; electrocardiography; interventricular heterogeneities; inward rectifier potassium current doi:10.1152/ajpheart.01312.2006.

Mechanisms underlying nutrient-induced segmentation within the gut are not well understood. We have shown that decanoic acid and some amino acids induce neurally dependent segmentation in guinea pig small intestine in vitro. This study examined the neural mechanisms underlying segmentation in the circular muscle and whether the timing of segmentation contractions also depends on slow waves. Decanoic acid (1 mM) was infused into the lumen of guinea pig duodenum and jejunum. Video imaging was used to monitor intestinal diameter as a function of both longitudinal position and time. Circular muscle electrical activity was recorded by using suction electrodes. Recordings from sites of segmenting contractions showed they are always associated with excitatory junction potentials leading to action potentials. Recordings from sites oral and anal to segmenting contractions revealed inhibitory junction potentials that were time locked to those contractions. Slow waves were never observed underlying segmenting contractions. In paralyzed preparations, intracellular recording revealed that slow-wave frequency was highly consistent at 19.5 (SD 1.4) cycles per minute (c/min) in duodenum and 16.6 (SD 1.1) c/min in jejunum. By contrast, the frequencies of segmenting contractions varied widely (duodenum: 3.6-28.8 c/min, median 10.8 c/min; jejunum: 3.0-27.0 c/min, median 7.8 c/min) and sometimes exceeded slow-wave frequencies for that region. Thus nutrient-induced segmentation contractions in guinea pig small intestine do not depend on slow-wave activity. Rather they result from a neural circuit producing rhythmic localized activity in excitatory motor neurons, while simultaneously activating surrounding inhibitory motor neurons. enteric nervous system; intestinal motility patterns; slow waves

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 ([PAC.sub.1]) 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. [[Ala.sup.4]]- and [[Val.sup.5]]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [[Ala.sup.4], [Val.sup.5]]- and [[Ala.sup.4], [Val.sup.5], [Asn.sup.9]]PACAP-27 induced relaxation similarly to VIP. [[Asn.sup.9]]-, [[Thr.sup.11]]-, or [[Leu.sup.13]]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [[Asn.sup.24], [Ser.sup.25], [Ile.sup.26]]PACAP-27 showed no change in the activity. All VIP analogs, including [[Gly.sup.4], [Ile.sup.5], [Ser.sup.9]] VIP, induced relaxation. In the presence of a [PAC.sub.1] receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of [PAC.sub.1] receptor, 'hop' splice variant, and [VPAC.sub.1] and [VPAC.sub.2] receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via [PAC.sub.1]/hop receptors. [Gly.sup.4] and [Ile.sup.5] are the key N[H.sub.2]-terminal residues of PACAP-27 that distinguish [PAC.sub.1]/hop receptors from [VPAC.sub.1]/[VPAC.sub.2] receptors. However, both the N[H.sub.2]-terminal and [alpha]-helical regions of PACAP-27 are required for initiating gallbladder contraction. [PAC.sub.1] receptor; [VPAC.sub.1] receptor; [VPAC.sub.2] receptor; splice variant

Earlier, we found that systemic administration of lipopolysaccharide (LPS; 4 mg/kg) hyperpolarized the transepithelial potential difference ([V.sub.t]) of tracheal epithelium in the isolated, perfused trachea (1PT) of the guinea pig 18 h after injection. As well, LPS increased the hyperpolarization component of the response to basolateral methacholine, and potentiated the epithelium-derived relaxing factor-mediated relaxation responses to hyperosmolar solutions applied to the apical membrane. We hypothesized that LPS stimulates the transepithelial movement of [Na.sup.+] via the epithelial sodium channel (ENaC)/[Na.sup.+]-[K.sup.+] pump axis, leading to hyperpolarization of [V.sub.t]. LPS increased the [V.sub.t]-depolarizing response to amiloride (10 [micro]M), i.e., offset the effect of LPS, indicating that [Na.sup.+] transport activity was increased. The functional activity of ENaC was measured in the IPT after short-circuiting the [Na.sup.+]-[K.sup.+] pump with basolateral amphotericin B (7.5 [micro]M). LPS had no effect on the hyperpolarization response to apical trypsin (100 U/ml) in the Ussing chamber, indicating that channel-activating proteases are not involved in the LPS-induced activation of ENaC. To assess [Na.sup.+]-[K.sup.+] pump activity in the IPT, ENaC was short-circuited with apical amphotericin B. The greater [V.sub.t] in the presence of amphotericin B in tracheas from LPS-treated animals compared with controls revealed that LPS increased [Na.sup.+]-[K.sup.+] pump activity. This finding was confirmed in the Ussing chamber by inhibiting the [Na.sup.+]-[K.sup.+] pump via extracellular [K.sup.+] removal, loading the epithelium with [Na.sup.+], and observing a greater hyperpolarization response to [K.sup.+] restoration. Together, the findings of this study reveal that LPS hyperpolarizes the airway epithelium by increasing the activities of ENaC and the [Na.sup.+]-[K.sup.+] pump. endotoxin; lung; airway epithelium; [Na.sup.+] transport; transepithelial voltage doi: 10.1152/ajplung.00123.2010.

Prior studies indicated that a [Ca.sup.2+]-dependent release of ATP can be initiated from the soma of sympathetic neurons dissociated from guinea pig stellate ganglia. Previous studies also indicated that [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) can modulate membrane excitability in these same neurons. As [Ca.sup.2+] release from internal stores is thought to support somatodendritic transmitter release in other neurons, the present study investigated whether CICR is essential for somatic ATP release from dissociated sympathetic neurons. Caffeine increased intracellular [Ca.sup.2+] and activated two inward currents: a slow inward current (SIC) in 85% of cells, and multiple faster inward currents [asynchronous transient inward currents (ASTICs)] in 40% of cells voltage-clamped to negative potentials. Caffeine evoked both currents when cells were bathed in a [Ca.sup.2+]-deficient solution, indicating that both were initiated by [Ca.sup.2+] release from ryanodine-sensitive stores in the endoplasmic reticulum. Sodium influx contributed to generation of both SICs and ASTICs, but only ASTICs were inhibited by the presence of the P2X receptor blocker PPADs. Thus ASTICs, but not SICs, resulted from an ATP activation of P2X receptors. Ionomycin induced ASTICs in a [Ca.sup.2+]-containing solution, but not when it was applied in a [Ca.sup.2+]-deficient solution, demonstrating the key requirement for external [Ca.sup.2+] in initiating ASTICs by ionomycin. Pretreatment with drugs to deplete the internal stores of [Ca.sup.2+] did not block the ability of ionomycin or long depolarizing voltage steps to initiate ASTICs. Although a caffeine-induced release of [Ca.sup.2+] from internal stores can elicit both SICs and ASTICs in dissociated sympathetic neurons, CICR is not required for the somatic release of ATP. nonclassical transmitter release; autonomic neurons; asynchronous transient inward currents; slow inward currents doi: 10.1152/ajpcell.00036.2010.

Adrenergic stimulation of isolated guinea pig distal colonic mucosa produced transient [Cl.sup.-] and sustained [K.sup.+] secretion. Transient short-circuit current ([I.sub.sc]) depended on [[beta].sub.2]-adrenergic receptors ([[beta].sub.2]-AdrR), and sustained [I.sub.sc] relies on a [[beta].sub.1]-AdrR/[[beta].sub.2]-AdrR complex. Epinephrine (epi) increased cAMP content with a biphasic time course similar to changes in epi-activated [I.sub.sc] ([sup.epi][I.sub.sc]). Inhibition of transmembrane adenylyl cyclases (tmACs) reduced peak [sup.epi][I.sub.sc] and cAMP to near zero without decreasing sustained [sup.epi][I.sub.sc], consistent with cAMP from tmAC signaling for only [Cl.sup.-] secretion. Inhibition of soluble adenylyl cyclase (sAC) reduced sustained [sup.epi][I.sub.sc] and cAMP to near zero without decreasing peak [sup.epi][I.sub.sc] or cAMP, consistent with cAMP from sAC signaling for [K.sup.+] secretion. Sensitivity to phosphodiesterase (PDE) inhibitors and peptide YY (PYY) stimulation further supported separate signaling for the two components. PDE3 or PDE4 inhibitors enhanced peak [sup.epi][I.sub.sc] but not sustained [sup.epi][I.sub.sc], consistent with these PDEs as part of the [[beta].sub.2]-AdrR signaling domain. PYY suppressed peak [sup.epi][I.sub.sc] in a pertussis toxin (PTx)-sensitive manner, supporting G[[alpha].sub.i]-dependent inhibition of tmACs producing cAMP for [Cl.sup.-] secretion. Since PYY or PTx did not alter sustained [sup.epi][I.sub.sc], signaling for [K.sup.+] secretion occurred via a G[[alpha].sub.i]-independent mechanism. Presence of multiple sAC variants in colonic epithelial cells was supported by domain-specific antibodies. Responses to specific activators and inhibitors suggested that protein kinase A was not involved in activating peak or sustained components of [sup.epi][I.sub.sc], but the cAMP-dependent guanine nucleotide exchange factor, Epac, may contribute. Thus [beta]-adrenergic activation of electrogenic [Cl.sup.-] and [K.sup.+] secretion, respectively, required tmAC- and sAC-dependent signaling pathways. epinephrine; norepinephrine; soluble adenylyl cyclase; protein kinase A; Epac doi: 10.1152/ajpgi.00035.2010.

The neuronal mechanism by which distension of the colon triggers peristalsis and the propulsion of colonic contents is incompletely understood. In this study, we used video imaging and spatiotemporal mapping techniques to investigate the neuroneuronal mechanisms underlying peristalsis in isolated guinea pig distal colon. In direct contrast to previous studies, we found that hexamethonium (100 [micro]M-1 mM) or mecamylamine (20 [micro]M) never abolished peristalsis or fecal pellet propulsion, although a temporary blockade of peristalsis was common, giving the impression perhaps that peristalsis was blocked permanently. During the initiation of peristalsis, the intraluminal propulsive force applied to an inserted fecal pellet was significantly reduced by hexamethonium 100 [micro]M, even though, once initiated, the propagation velocity of fecal pellets was never reduced by nicotinic antagonists. In the presence of hexamethonium or mecamylamine, further addition of PPADS (10 [micro]M), ondansetron (1 [micro]M), and SR 142801 (300 nM) had no inhibitory effect on the propagation velocity of fecal pellets. In these preparations, antagonists for nicotinic, purinergic (P2), serotonergic (5-HT3), or tachykinergic (NK3) receptors always abolished responses to the agonists for these receptors, confirming that when peristalsis occurred, nicotinic, P2, 5-HT3, and NK3 receptors were blocked. Tetrodotoxin abolished nonnicotinic peristalsis. In summary, nicotinic transmission contributes to excitatory neuroneuronal transmission underlying peristalsis and fecal pellet propulsion but is not required for peristalsis, nor fecal pellet propulsion, as once thought. These observations could be explained by an excitatory nonnicotinic neuroneuronal pathway that can generate peristalsis and induce normal fecal pellet propagation velocities but does not require nicotinic, P2, 5-HT3, or NK3 receptors. sensory neuron; enteric; reflex; mechanosensory; afferent; synaptic doi: 10.1152/ajpgi.00457.2009.

Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by [[alpha].sub.2]-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RTPCR, and confocal microscopy to test whether [5-HT.sub.1A], [SST.sub.1], and/or [SST.sub.2] receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific [5-HT.sub.1A] receptor antagonist WAY 100135 (1 [micro]M) reduced the amplitude of IPSPs, an effect that persisted in the presence of the [[alpha].sub.2]-adrenoceptor antagonist idazoxan (2 [micro]M), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific [SSTR.sub.2] antagonist CYN 154806 (100 nM) and a specific [SSTR.sub.1] antagonist SRA 880 (3 [micro]M) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for [5-HT.sub.1A], [SST.sub.1], and [SST.sub.2] receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that [5-HT.sub.1A], [SST.sub.1], and [SST.sub.2] receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes. somatostatin; serotonin; secretomotor neurons; inhibitory interneurons; nonadrenergic inhibitory synaptic potentials doi:10.1152/ajpgi.00438.2009.

Patterns of salivary [HCO.sup.-.sub.3] secretion vary and depend on species and gland types. However, the identities of the transporters involved in [HCO.sup.-.sub.3] transport and the underlying mechanism of intracellular pH ([pH.sub.i]) regulation in salivary glands still remain unclear. In this study, we examined the expression of the [Na.sup.+]-[HCO.sup.-.sub.3] cotransporter (NBC) and its role in [pH.sub.i] regulation in guinea pig salivary glands, which can serve as an experimental model to study [HCO.sup.-.sub.3] transport in human salivary glands. RT-PCR, immunohistochemistry, and [pH.sub.i] measurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive [Na.sup.+]/[H.sup.+] exchanger (NHE) played a putative role in [pH.sub.i] regulation in salivary acinar cells and also appeared to be involved in regulation in salivary ducts. In addition to NHE, NBC also played a role in [pH.sub.i] regulation in both acini and ducts. In the parotid gland, NBC1 was functionally expressed in the basolateral membrane (BLM) of acinar cells and the luminal membrane (LM) of ducts. In the submandibular gland, NBC1 was expressed only in the BLM of ducts. NBC1 expressed in these two types of salivary glands takes up [HCO.sup.-.sub.3] and is involved in [pH.sub.i] regulation. Although NBC3 immunoreactivity was also detected in submandibular gland acinar cells and in the ducts of both glands, it is unlikely that NBC3 plays any role in [pH.sub.i] regulation. We conclude that NBC1 is functionally expressed and plays a role in [pH.sub.i] regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands. intracellular pH; [Na.sup.+]/[H.sup.+] exchange

The transient outward current ([I.sub.to]) is a major repolarizing current in the heart. Marked reduction of Ito density occurs in heart failure and is accompanied by significant action potential duration (APD) prolongation. To understand the species-dependent role of [I.sub.to] in regulating the ventricular action potential morphology and duration, we introduced simulated [I.sub.to] conductance in guinea pig and canine endocardial ventricular myocytes using the dynamic clamp technique and perforated patch-clamp recordings. The effects of simulated [I.sub.to] in both types of cells were complex and biphasic, separated by a clear density threshold of ~40 pA/pF. Below this threshold, simulated [I.sub.to] resulted in a distinct phase 1 notch and had little effect on or moderately prolonged the APD. [I.sub.to] above the threshold resulted in all-or-none repolarization and precipitously reduced the APD. Qualitatively, these results agreed with our previous studies in canine ventricular cells using whole cell recordings. We conclude that 1) contrary to previous gene transfer studies involving the Kv4.3 current, the response of guinea pig ventricular myocytes to a fully inactivating [I.sub.to] is similar to that of canine ventricular cells and 2) in animals such as dogs that have a broad cardiac action potential, [I.sub.to] does not play a major role in setting the APD. dynamic clamp; transient outward current; ventricular myocytes doi:10.1152/ajpheart.00084.2006

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal [Ca.sup.2+] imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine [10.sup.-6] M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM [K.sup.+]-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF [10.sup.-6] M and CRF [10.sup.-7] M during 30 s induced a [Ca.sup.2+] response in 22.4% of the myenteric neurons (n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin (n = 55). The selective CRF-1 receptor antagonist CP 154,526 (n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at [10.sup.-7] M, and its effect at [10.sup.-6] M (15.3%, n = 59) was completely blocked by CP 154,526. TTX [10.sup.-6] M (n = 70) could not block the CRF-induced [Ca.sup.2+] transients but reduced the amplitude of the signals significantly. Removal of extracellular [Ca.sup.2+] blocked all responses to CRF (n = 47). L-type channels did not contribute to the CRF-induced [Ca.sup.2+] transients. Blocking N- or P/Q-type [Ca.sup.2+] channels did not reduce the responses significantly. Combined L- and R-type [Ca.sup.2+] channel blocking (SNX-482 [10.sup.-8] M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces [Ca.sup.2+] transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These [Ca.sup.2+] transients highly depend on somatic calcium influx through voltage-operated [Ca.sup.2+] channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the [Ca.sup.2+] signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level. corticotropin-releasing factor; calcium imaging; FLUO-4; intestinal motility; confocal microscopy; R-type voltage-sensitive [Ca.sup.2+] channels

In guinea pig antral mucous cells, ACh stimulates the [Ca.sup.2+]-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of cGMP on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. cGMP enhanced the frequency of ACh-stimulated exocytotic events, whereas cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition, cGMP did not stimulate either [Ca.sup.2+] mobilization or cAMP accumulation. The [Ca.sup.2+] dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower concentration. This indicates that cGMP increased maximal responsiveness of the [Ca.sup.2+]-regulated exocytosis, but not the [Ca.sup.2+] sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia ([N.sub.2] bubbling), ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of [Ca.sup.2+]-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the [Ca.sup.2+]-regulated exocytosis stimulated by ACh. gastric mucin secretion; guanosine 3'-cyclic monophosphate; exocytosis; acetylcholine; intracellular calcium concentration

The effect of [beta]-adrenergic stimulation on cardiac N[a.sup.+]/C[a.sup.2+] exchange has been controversial. To clarify the effect, we measured N[a.sup.+]/C[a.sup.2+] exchange current ([I.sub.NCX)] in voltage-clamped guinea pig, mouse, and rat ventricular cells. When [I.sub.NCX] was defined as a 5 mM N[i.sup.2+]-sensitive current in guinea pig ventricular myocytes, 1 [micro]M isoproterenol apparently augmented [I.sub.NCX] by ~32%. However, this increase was probably due to contamination of the cAMP-dependent C[l.sup.-] current (CFTR-C[l.sup.-] current, [I.sub.CFTR-Cl]), because N[i.sup.2+] inhibited the activation of [I.sub.CFTR-Cl] by 1 [micro]M isoproterenol with a half-maximum concentration of 0.5 mM under conditions where [I.sub.NCX] was suppressed. Five or ten millimolar N[i.sup.2+] did not inhibit [I.sub.CFTR-Cl] activated by 10 [micro]M forskolin, an activator of adenylate cyclase, suggesting that N[i.sup.2+] acted upstream of adenylate cyclase in the [beta]-adrenergic signaling pathway. Furthermore, in a low-extracellular C[l.sup.-] bath solution, 1 [micro]M isoproterenol did not significantly alter the amplitude of N[i.sup.2+]-sensitive [I.sub.NCX] at +50 mV, which is close to the reversal potential of [I.sub.CFTR-Cl]. NO change in [I.sub.NCX] amplitude was induced by 10 [micro]M forskolin. When [I.sub.NCX] was activated by extracellular C[a.sup.2+], it was not significantly affected by 1 [micro]M isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that [beta]-adrenergic stimulation does not have significant effects on [I.sub.NCX] in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion

Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted ~20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a twofold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators. intrinsic cardiac nervous system; histamine; pituitary adenylate cyclase-activating polypeptide; mast cells; nitric oxide synthase; intracellular recording

We investigated the effect of inflammation on slow synaptic transmission in myenteric neurons in the guinea pig ileum. Inflammation was induced by the intraluminal injection of trinitrobenzene sulfonate, and tissues were taken for in vitro investigation 6-7 days later. Brief tetanic stimulation of synaptic inputs (20 Hz, 1 s) induced slow excitatory postsynaptic potentials (EPSPs) in 49% and maintained postsynaptic excitation that lasted from 27 min to 3 h in 13% of neurons from the inflamed ileum. These neurons were classified electrophysiologically as AH neurons; 10 were morphological type II neurons, and one was type I. Such long-term hyperexcitability after a brief stimulus is not encountered in enteric neurons of normal intestine. Electrophysiological properties of neurons with maintained postsynaptic excitation were similar to those of neurons with slow EPSPs. Another form of prolonged excitation, sustained slow postsynaptic excitation (SSPE), induced by 1-Hz, 4-min stimulation, in type II neurons from the inflamed ileum reached its peak earlier but had lower amplitude than that in control. Unlike slow EPSPs and similar to SSPEs, maintained excitation was not inhibited by neurokinin-1 or neurokinin-3 receptor antagonists. Maintained postsynaptic excitation was not influenced by PKC inhibitors, but the PKA inhibitor, H-89, caused further increase in neuronal excitability. In conclusion, maintained excitation, observed only in neurons from the inflamed ileum, may contribute to the dysmotility, pain, and discomfort associated with intestinal inflammation. enteric nervous system; myenteric neurons

Adrenergic stimulation of electrogenic [K.sup.+] secretion in isolated mucosa from guinea pig distal colon required activation of two [beta]-adrenergic receptor subtypes (13-AdrR). Addition of epinephrine (epi) or norepinephrine (norepi) to the bathing solution of mucosae in Ussing chambers increased short-circuit current ([I.sub.sc]) and transepithelial conductance ([G.sub.t]), consistent with this cation secretion. A [beta]-adrenergic classification was supported by propranolol antagonism of this secretory response and the lack of effect by the [alpha]-AdrR antagonists BE2254 ([alpha]l-AdrR) and yohimbine ([alpha]2-AdrR). Subtype-selective antagonists CGP20712A ([beta]1-AdrR), ICI-118551 ([beta]2-AdrR), and SR59320A ([beta]3-AdrR) were relatively ineffective at inhibiting the epi-stimulated [I.sub.sc] response. In combination, CGP20712A and ICI-118551 inhibited the response, which supported a synergistic action by [beta]1-AdrR and [beta]2-AdrR. Expression of mRNA for both [beta]l-AdrR and [beta]2-AdrR was indicated by RT-PCR of RNA from colonic epithelial cells. Protein expression was indicated by immunoblot showing bands at molecular weights consistent with monomers and oligomers. Immunoreactivity (ir) for [beta]1-AdrR and [beta]2-AdrR was prominent in basolateral membranes of columnar epithelial cells in the crypts of Lieberkahn as well as intercrypt surface epithelium. Cells in the pericryptal sheath also had [beta]1-Adr[R.sup.ir] but did not have discernable [beta]2-Adr[R.sup.ir]. The adrenergic sensitivity of [K.sup.+] secretion measured by [I.sub.sc] and [G.sub.t] was relatively low as indicated by [EC.sub.50]s of 41 [+ or -] 7 nM for epi and 50 [+ or -] 14 nM for norepi. Adrenergic activation of electrogenic [K.sup.+] secretion required the involvement of both [beta]1-AdrR and [beta]2-AdrR, occurring with an agonist sensitivity reduced compared with reported values for either receptor subtype. epinephrine; norepinephrine; isoproteranol; CGP20712A; ICI-118551

Adrenergic activation of electrogenic [K.sup.+] secretion in isolated mucosa from guinea pig distal colon was desensitized by peptide-YY (PYY). Addition of PYY or neuropeptide-Y (NPY) to the bathing solution of mucosae in Ussing chambers suppressed the short-circuit current ([I.sub.sc]) corresponding to electrogenic [Cl.sup.-] secretion, whether stimulated by epinephrine (epi), prostaglandin-[E.sub.2] ([PGE.sub.2]), or carbachol (CCh). Neither peptide markedly inhibited the large transient component of synergistic secretion ([PGE.sub.2] + CCh). Sustained [Cl.sup.-] secretory [I.sub.sc] was inhibited ~65% by PYY or NPY, with [IC.sub.50]s of 4.1 [+ or -] 0.9 nM and 9.4 [+ or -] 3.8 nM, respectively. This inhibition was eliminated by BIIE0246, an antagonist of the Y2-neuropeptide receptor (Y2-NpR), but not by Y I-NpR antagonist BVD 10. Adrenergic sensitivity for activation of [K.sup.+] secretion in the presence of Y2-NpR blockade by BIIE0246 was ([IC.sub.50]s) 2.9 [+ or -] 1.2 nM for epi and 13.3 [+ or -] 1.0 nM for norepinephrine, approximately fourfold greater than in the presence of PYY. Expression of mRNA for both Y1-NpR and Y2-NpR was indicated by RT-PCR of RNA from colonic mucosa, and protein expression was indicated by immunoblot. Immunoreactivity (ir) for Y1-NpR and Y2-NpR was distinct in basolateral membranes of columnar epithelial cells in the crypts of Lieberktihn as well as intercrypt surface epithelium. Adrenergic nerves in proximity with crypts were detected by ir for dopamine-[beta]-hydroxylase, and a portion of these nerves also contained [NPY.sup.ir]. BIIE0246 addition increased secretagog-activated [I.sub.sc], consistent with in vitro release of either PYY or NPY. Thus PYY and NPY were able to suppress [Cl.sup.-] secretory capacity and desensitize the adrenergic [K.sup.+] secretory response, providing a direct inhibitory counterbalance against secretory activation. epinephrine; norepinephrine; ATP; adenosine; Y1-neuropeptide receptor; enteric nerves; [beta]-adrenergic receptor; [beta]2-adrenergic receptor