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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.

Multiple types of Cl− channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl− channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl− channels in freshly isolated guinea pig DSM cells. The recorded single Cl− channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of −41.5 mV, which is close to the ECl of −65 mV, confirming preferential permeability to Cl−. The Cl− channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V0.5, ~−20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca2+-free or 1 mM Ca2+. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl− with I−, Br−, or methanesulfonate (MsO−), resulting in anionic permeability sequence: Cl−>Br−>I−>MsO−. While intracellular Ca2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl− current and outward rectification, high Ca2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca2+-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl− channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

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.

This study addressed the hypothesis that long-term deficiency of ovarian hormones after ovariectomy (OVx) alters cellular Ca2+-handling mechanisms in the heart, resulting in the formation of a proarrhythmic substrate. It also tested whether estrogen supplementation to OVx animals reverses any alterations to cardiac Ca2+ handling and rescues proarrhythmic behavior. OVx or sham operations were performed on female guinea pigs using appropriate anesthetic and analgesic regimes. Pellets containing 17β-estradiol (1 mg, 60-day release) were placed subcutaneously in selected OVx animals (OVx + E). Cardiac myocytes were enzymatically isolated, and electrophysiological measurements were conducted with a switch-clamp system. In fluo-4-loaded cells, Ca2+ transients were 20% larger, and fractional sarcoplasmic reticulum (SR) Ca2+ release was 7% greater in the OVx group compared with the sham group. Peak L-type Ca2+ current was 16% larger in OVx myocytes with channel inactivation shifting to more positive membrane potentials, creating a larger “window” current. SR Ca2+ stores were 22% greater in the OVx group, and these cells showed a higher frequency of Ca2+ sparks and waves and shorter wave-free intervals. OVx myocytes showed higher frequencies of early afterdepolarizations, and a greater percentage of these cells showed delayed afterdepolarizations after exposure to isoprenaline compared with sham myocytes. The altered Ca2+ regulation occurring in the OVx group was not observed in the OVx + E group. These findings suggest that long-term deprivation of ovarian hormones in guinea pigs lead to changes in myocyte Ca2+-handling mechanisms that are considered proarrhythmogenic. 17β-Estradiol replacement prevented these adverse effects. NEW & NOTEWORTHY Ovariectomized guinea pig cardiomyocytes have higher frequencies of Ca2+ waves, and isoprenaline-challenged cells display more early afterdepolarizations, delayed afterdepolarizations, and extra beats compared with sham myocytes. These alterations to Ca2+ regulation were not observed in myocytes from ovariectomized guinea pigs supplemented with 17β-estradiol, suggesting that ovarian hormone deficiency modifies cardiac Ca2+ regulation, potentially creating proarrhythmic substrates.

Mitochondria are in close proximity to the redox-sensitive sarcoplasmic reticulum (SR) Ca2+ release [ryanodine receptors (RyRs)] and uptake [Ca2+-ATPase (SERCA)] channels. Thus mitochondria-derived reactive oxygen species (mdROS) could play a crucial role in modulating Ca2+ cycling in the cardiomyocytes. However, whether mdROS-mediated Ca2+ dysregulation translates to abnormal electrical activities under pathological conditions, and if yes what are the underlying ionic mechanisms, have not been fully elucidated. We hypothesize that pathological mdROS induce Ca2+ elevation by modulating SR Ca2+ handling, which activates other Ca2+ channels and further exacerbates Ca2+ dysregulation, leading to abnormal action potential (AP). We also propose that the morphologies of elicited AP abnormality rely on the time of mdROS induction, interaction between mitochondria and SR, and intensity of mitochondrial oxidative stress. To test the hypotheses, we developed a multiscale guinea pig cardiomyocyte model that incorporates excitation-contraction coupling, local Ca2+ control, mitochondrial energetics, and ROS-induced ROS release. This model, for the first time, includes mitochondria-SR microdomain and modulations of mdROS on RyR and SERCA activities. Simulations show that mdROS bursts increase cytosolic Ca2+ by stimulating RyRs and inhibiting SERCA, which activates the Na+/Ca2+ exchanger, Ca2+-sensitive nonspecific cationic channels, and Ca2+-induced Ca2+ release, eliciting abnormal AP. The morphologies of AP abnormality are largely influenced by the time interval among mdROS burst induction and AP firing, dosage and diffusion of mdROS, and SR-mitochondria distance. This study defines the role of mdROS in Ca2+ overload-mediated cardiac arrhythmogenesis and underscores the importance of considering mitochondrial targets in designing new antiarrhythmic therapies.

Cl−channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl−conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl−current in freshly isolated guinea pig DSM cells. The Cl−current demonstrated outward rectification arising from voltage-dependent gating of Cl−channels rather than the Cl−transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl−current providing strong evidence that volume-regulated anion channels do not contribute to the Cl−current in DSM cells. The Cl−current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] analog [PI(4,5)P2-diC8] increased the average Cl−current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl−current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P2-diC8, an intriguing finding that underscores the complex nature of Cl−channel expression and function in DSM cells. Removal of both extracellular Ca2+and Mg2+did not affect the DSM whole cell Cl−current, whereas Gd3+(1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl−channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd3+, and PI(4,5)P2.

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5–10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels ( Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca2+-regulated exocytosis, consisting of a peak in exocytotic events that declines rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation. GW7647 [a peroxisome proliferation activation receptor α (PPARα) agonist] enhanced the ACh-stimulated initial phase, and GW6471 (a PPARα antagonist) abolished the GW7647-induced enhancement. However, GW6471 produced the delayed, but transient, increase in the ACh-stimulated late phase, and it also decreased the initial phase and produced the delayed increase in the late phase during stimulation with ACh alone. A similar delayed increase in the ACh-stimulated late phase is induced by an inhibitor of the PKG, Rp8BrPETcGMPS, suggesting that GW6471 inhibits cGMP accumulation. An inhibitor of nitric oxide synthase 1 (NOS1), N5-[imino(propylamino)methyl]-l-ornithine hydrochloride ( N-PLA), also abolished the GW7647-induced-enhancement of ACh-stimulated initial phase but produced the delayed increase in the late phase. However, in the presence of N-PLA, an NO donor or 8BrcGMP enhanced the ACh-stimulated initial phase and abolished the delayed increase in the late phase. Moreover, GW7647 and ACh stimulated NO production and cGMP accumulation in antral mucosae, which was inhibited by GW6471 or N-PLA. Western blotting and immunohistochemistry revealed that NOS1 and PPARα colocalize in antral mucous cells. In conclusion, during ACh stimulation, a PPARα autocrine mechanism, which accumulates NO via NOS1 leading to cGMP accumulation, modulates the Ca2+-regulated exocytosis in antral mucous cells.

Spatiotemporal changes in cytosolic Ca2+ concentration ([Ca2+]c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca2+-induced Ca2+ release following membrane depolarization as local Ca2+ transients, Ca2+ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca2+ signaling was examined. Cytosolic and mitochondrial Ca2+ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca2+]c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca2+ concentration in the mitochondria matrix ([Ca2+]m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca2+]c returned to the resting level with a half-decay time of ~500 ms, while [Ca2+]m recovered more slowly (∼1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca2+]m elevations and slowed [Ca2+]c changes. Importantly, short depolarization-induced changes in [Ca2+]m and transmembrane potential in mitochondria coupled to Ca2+ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca2+ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca2+ microdomain, and thus, strongly contribute to [Ca2+]c regulation in SMCs.

The question of the extent to which cytosolic Ca2+ affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the “Ca2+ clock” (C) and “membrane clock” (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na+/Ca2+ exchange current via spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca2+, whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ∼30 s, whereas contraction ceased within ∼3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca2+ dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca2+ dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca2+-mediated inactivation of the L-type Ca2+ currents induced by BAPTA, were well explained by assuming Ca2+ chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca2+ current rundown induced secondarily through inhibition of Ca2+/calmodulin kinase II and/or Ca2+-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca2+ concentration reduction for >5 min.

Glucagon-like peptide-1 (GLP-1) acts at the G protein-coupled receptor, GLP-1R, to stimulate secretion of insulin and to inhibit secretion of glucagon and gastric acid. Involvement in mucosal secretory physiology has received negligible attention. We aimed to study involvement of GLP-1 in mucosal chloride secretion in the small intestine. Ussing chamber methods, in concert with transmural electrical field stimulation (EFS), were used to study actions on neurogenic chloride secretion. ELISA was used to study GLP-1R effects on neural release of acetylcholine (ACh). Intramural localization of GLP-1R was assessed with immunohistochemistry. Application of GLP-1 to serosal or mucosal sides of flat-sheet preparations in Ussing chambers did not change baseline short-circuit current ( Isc), which served as a marker for chloride secretion. Transmural EFS evoked neurally mediated biphasic increases in Iscthat had an initial spike-like rising phase followed by a sustained plateau-like phase. Blockade of the EFS-evoked responses by tetrodotoxin indicated that the responses were neurally mediated. Application of GLP-1 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-1 receptor antagonist exendin-(9–39) suppressed this action of GLP-1. The GLP-1 inhibitory action on EFS-evoked responses persisted in the presence of nicotinic or vasoactive intestinal peptide receptor antagonists but not in the presence of a muscarinic receptor antagonist. GLP-1 significantly reduced EFS-evoked ACh release. In the submucosal plexus, GLP-1R immunoreactivity (IR) was expressed by choline acetyltransferase-IR neurons, neuropeptide Y-IR neurons, somatostatin-IR neurons, and vasoactive intestinal peptide-IR neurons. Our results suggest that GLP-1R is expressed in guinea pig submucosal neurons and that its activation leads to a decrease in neurally evoked chloride secretion by suppressing release of ACh at neuroepithelial junctions in the enteric neural networks that control secretomotor functions.

Adeno-associated virus delivery systems and short hairpin RNA (shRNA) were used to selectively silence the voltage-gated sodium channel NaV 1.7 in the nodose ganglia of guinea pigs. The cough reflex in these animals was subsequently assessed. NaV 1.7 shRNA was delivered to the majority of nodose ganglia neurons [50–60% transfection rate determined by green fluorescent protein (GFP) gene cotransfection] and action potential conduction in the nodose vagal nerve fibers, as evaluated using an extracellular recording technique, was markedly and significantly reduced. By contrast, V 1.7 silencing in the nodose ganglia nearly abolished cough evoked by mechanically probing the tracheal mucosa in anesthetized guinea pigs. Stimuli such as capsaicin and bradykinin that are known to stimulate both nodose and jugular C-fibers evoked coughing in conscious animals was unaffected by NaV 1.7 silencing in the nodose ganglia. Nodose C-fiber selective stimuli including adenosine, 2-methyl-5-HT, and ATP all failed to evoke coughing upon aerosol challenge. These results indicate that cough is independently regulated by two vagal afferent nerve subtypes in guinea pigs, with nodose Aδ fibers regulating cough evoked mechanically from the trachea and bradykinin- and capsaicin-evoked cough regulated by C-fibers arising from the jugular ganglia.

Although β2-adrenoceptors represent 15–25% of β-adrenoceptors in the guinea pig heart, their functionality is controversial. We assessed the inotropic effects of β2-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 μM) due to activation of β1-adrenoceptors. In the presence of 4 μM atenolol, the concentration-dependent NIE (−12% at 6 μM) was biphasic, best described by a double logistic equation with respective EC50 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 A2 (cPLA2) 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 cPLA2. The possibility that these effects are due to an equilibrium between different affinity states of the receptor (Gs/Gi coupled and Gi independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that β2-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state.

Earlier, we found that systemic administration of lipopolysaccharide (LPS; 4 mg/kg) hyperpolarized the transepithelial potential difference (Vt) of tracheal epithelium in the isolated, perfused trachea (IPT) 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+ via the epithelial sodium channel (ENaC)/Na+-K+ pump axis, leading to hyperpolarization of Vt. LPS increased the Vt-depolarizing response to amiloride (10 μM), i.e., offset the effect of LPS, indicating that Na+ transport activity was increased. The functional activity of ENaC was measured in the IPT after short-circuiting the Na+-K+ pump with basolateral amphotericin B (7.5 μM). 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+-K+ pump activity in the IPT, ENaC was short-circuited with apical amphotericin B. The greater Vt in the presence of amphotericin B in tracheas from LPS-treated animals compared with controls revealed that LPS increased Na+-K+ pump activity. This finding was confirmed in the Ussing chamber by inhibiting the Na+-K+ pump via extracellular K+ removal, loading the epithelium with Na+, and observing a greater hyperpolarization response to K+ restoration. Together, the findings of this study reveal that LPS hyperpolarizes the airway epithelium by increasing the activities of ENaC and the Na+-K+ pump.

Bradykinin (BK) activates sensory nerves and causes hyperalgesia. Transient receptor potential A1 (TRPA1) is expressed in sensory nerves and mediates cold, mechanical, and chemical nociception. TRPA1 can be activated by BK. TRPA1 knockout mice show impaired responses to BK and mechanical nociception. However, direct evidence from sensory nerve terminals is lacking. This study aims to determine the role of TRPA1 in BK-induced visceral mechanical hypersensitivity. Extracellular recordings of action potentials from vagal nodose and jugular neurons are performed in an ex vivo guinea pig esophageal-vagal preparation. Peak frequencies of action potentials of afferent nerves evoked by esophageal distension and chemical perfusion are recorded and compared. BK activates most nodose and all jugular C fibers. This activation is repeatable and associated with a significant increase in response to esophageal distension, which can be prevented by the B2 receptor antagonist WIN64338. TRPA1 agonist allyl isothiocyanate (AITC) activates most BK-positive nodose and jugular C fibers. This is associated with a transient loss of response to mechanical distensions and desensitization to a second AITC perfusion. Desensitization with AITC and pretreatment with TRPA1 inhibitor HC-030031 both inhibit BK-induced mechanical hypersensitivity but do not affect BK-evoked activation in nodose and jugular C fibers. In contrast, esophageal vagal afferent Aδ fibers do not respond to BK or AITC and fail to show mechanical hypersensitivity after BK perfusion. This provides the first evidence directly from visceral sensory afferent nerve terminals that TRPA1 mediates BK-induced mechanical hypersensitivity. This reveals a novel mechanism of visceral peripheral sensitization.

Eosinophilic esophagitis is characterized by increased infiltration and degranulation of eosinophils in the esophagus. Whether eosinophil-derived cationic proteins regulate esophageal sensory nerve function is still unknown. Using synthetic cationic protein to investigate such effect, we performed extracellular recordings from vagal nodose or jugular neurons in ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Nerve excitabilities were determined by comparing action potentials evoked by esophageal distensions before and after perfusion of synthetic cationic protein poly-l-lysine (PLL) with or without pretreatment with poly-l-glutamic acid (PLGA), which neutralized cationic charges of PLL. Perfusion with PLL did not evoke action potentials in esophageal nodose C fibers but increased their responses to esophageal distension. This potentiation effect lasted for 30 min after washing out of PLL. Pretreatment with PLGA significantly inhibited PLL-induced mechanohyperexcitability of esophageal nodose C fibers. In esophageal nodose Aδ fibers, perfusion with PLL did not evoke action potentials. In contrast to nodose C fibers, both the spontaneous discharges and the responses to esophageal distension in nodose Aδ fibers were decreased by perfusion with PLL, which can be restored after washing out PLL for 30–60 min. Pretreatment with PLGA attenuated PLL-induced decrease in spontaneous discharge and mechanoexcitability of esophageal nodose Aδ fibers. In esophageal jugular C fibers, PLL neither evoked action potentials nor changed their responses to esophageal distension. Collectively, these data demonstrated that synthetic cationic protein did not evoke action potential discharges of esophageal vagal afferents but had distinctive sensitization effects on their responses to esophageal distension.

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7–9 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 configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca2+imaging, and pharmacological agents. In our investigation of the role of intracellular Ca2+released from Ca2+stores, we observed that intracellular Ca2+transients accompanied some STDs, although there were many exceptions where Ca2+transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs) but not by treatments known to alter Ca2+release via ryanodine receptors. A role for Ca2+-activated Cl−(ClCa) channels was indicated, as STDs were dependent on the Cl−but not Na+concentration of the superfusing solution and were inhibited by the ClCachannel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl−blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd3+, and Ni2+had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd3+, and Ni2+altered the occurrence of spontaneous APs. Our findings support a role for Ca2+release through IP3Rs and a resultant opening of ClCachannels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.

To investigate how intercellular coupling can be changed during Ca2+ overloading of ventricular muscle, we studied Ca2+ signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential Ca2+ images of surface cells were obtained with a confocal microscope. In intact muscles, all cells showed simultaneous Ca2+ transients in response to field stimulation over a field of view of 0.3 × 0.3 mm2. In severely Ca2+-overloaded muscles, obtained by high-frequency stimulation in nonflowing Krebs solution, cells became less responsive to stimulation. Furthermore, nonsimultaneous but serial onsets of Ca2+ transients were often detected, suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in Ca2+-overloaded muscles, the distributions of phosphorylated and nonphosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the nonoverloaded muscles, whereas nonphosphorylated Cx43 was significantly elevated in severely Ca2+-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few square millimeters, can be a cause of abnormal conduction and a microreentry in Ca2+-overloaded heart. Inactivation of Na+ channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay.

Salicylate causes a moderate hearing loss and tinnitus in humans at high-dose levels. Salicylate-induced hearing loss has been attributed to impaired sound amplification by outer hair cells (OHCs) through its direct action on the OHC motility sensor and/or motor. However, there is a disparity of salicylate concentrations between the clinical and animal studies, i.e., extremely high extracellular concentrations of salicylate (from 1 to 10 mM) is required to produce a significant reduction of electromotility in animal studies. Such concentrations are above the clinical/physiological range for humans. Here, we showed that clinical/physiological concentration range of salicylate caused concentration-dependent and reversible reductions in IK,n (KCNQ4) and subsequent depolarization of OHCs. Salicylate reduced the maximal tail current of the activation curve of IK,n without altering the voltage-sensitivity ( Vhalf). The salicylate-induced reduction of IK,n was almost completely blocked by linopirdine (0.1 mM) and BaCl2 (10 mM). Consistent with the finding in OHCs, salicylate significantly reduced KCNQ4-mediated current expressed in Chinese hamster ovarian (CHO) cells by comparable amplitude to OHCs without significantly shifting Vhalf. Nonstationary fluctuation analysis shows that salicylate significantly reduced the estimated single-channel current amplitude and numbers. Intracellular Ca2+ elevation resulting from cytoplasmic acidosis also contributes to the current reduction of IK,n (KCNQ4) of OHCs. These results indicate a different model for the salicylate-induced hearing loss through the reduction of KCNQ4 and subsequent depolarization of OHCs, which reduces the driving force for transduction current and electromotility. The major mechanism underlying the reduction of IK,n (KCNQ4) is the direct blocking action of salicylate on KCNQ4.

Several esophageal pathologies are associated with an increased number of mast cells in the esophageal wall. We addressed the hypothesis that activation of esophageal mast cells leads to an increase in the excitability of local sensory C fibers. Guinea pigs were actively sensitized to ovalbumin. The mast cells in the esophagus were selectively activated ex vivo by superfusion with ovalbumin. Action potential discharge in guinea pig vagal nodose esophageal C-fiber nerve endings was monitored in the isolated (ex vivo) vagally innervated esophagus by extracellular recordings. Ovalbumin activated esophageal mast cells, leading to the rapid release of ∼20% of the tissue histamine stores. This was associated with a consistent and significant increase in excitability of the nodose C fibers as reflected in a two- to threefold increase in action potential discharge frequency evoked by mechanical (increases in intraluminal pressure) stimulation. The increase in excitability persisted unchanged for at least 90 min (longest time period tested) after ovalbumin was washed from the tissue. This effect could be prevented by the histamine H1 receptor antagonist pyrilamine, but once the increase in excitability occurred, it persisted in the nominal absence of histamine and could not be reversed even with large concentrations of the histamine receptor antagonist. In conclusion, activation of esophageal mast cells leads to a pronounced and long-lived increase in nociceptive C-fiber excitability such that any sensation or reflex evoked via the vagal nociceptors will likely be enhanced. The effect is initiated by histamine acting via H1 receptor activation and maintained in the absence of the initiating stimulus.

The incidence of urinary bladder disturbances increases with age, and free radical accumulation has been proposed as a causal factor. Here we investigated the association between changes in bladder neuromuscular function and oxidative stress in aging and the possible benefits of melatonin treatment. Neuromuscular function was assessed by electrical field stimulation (EFS) of isolated guinea pig detrusor strips from adult and aged female guinea pigs. A group of adult and aged animals were treated with 2.5 mg·kg−1·day−1 melatonin for 28 days. Neurotransmitter blockers were used to dissect pharmacologically the EFS-elicited contractile response. EFS induced a neurogenic and frequency-dependent contraction that was impaired by aging. This impairment is in part related to a decrease in detrusor myogenic contractility. Age also decreased the sensitivity of the contraction to pharmacological blockade of purinergic and sensitive fibers but increased the effect of blockade of nitrergic and adrenergic nerves. The density of cholinergic and nitrergic nerves remained unaltered, but aging modified afferent fibers. These changes were associated with an increased level of markers for oxidative stress. Melatonin treatment normalized oxidative levels and counteracted the aging-associated changes in bladder neuromuscular function. In conclusion, these results show that aging modifies neurogenic contraction and the functional profile of the urinary bladder plexus and simultaneously increases the oxidative damage to the organ. Melatonin reduces oxidative stress and improves the age-induced changes in bladder neuromuscular function, which could be of importance in reducing the impact of age-related bladder disorders.

Altered Na+/Ca2+ exchanger (NCX) protein expression or activity is thought to contribute to various aspects of cardiac pathology. In guinea pig ventricular myocytes, NCX-mediated Ca2+ entry is almost entirely responsible for Ca2+ overload during hypoxia-reoxygenation. Because Ca2+ overload is a common initiator of apoptosis, the purpose of this study was to test the hypotheses that NCX activity is critically involved in initiating apoptosis after hypoxia-reoxygenation and that hypoxia-reoxygenation-induced apoptosis can be modulated by changes in NCX protein expression or activity. An NCX antisense oligonucleotide was used to reduce NCX protein expression in cultured adult guinea pig ventricular myocytes. Caspase-3 activation and cytochrome c release were used as markers of apoptosis. Hypoxia-reoxygenation-induced apoptosis was significantly decreased in antisense-treated myocytes compared with untreated control or nonsense-treated myocytes. Pretreatment of cultured myocytes for 24 h with either endothelin-1 or phenylephrine was found to increase both NCX protein expression and evoked NCX activity as well as enhance hypoxia-reoxygenation-induced apoptosis. Control experiments demonstrated that endothelin-1 and phenylephrine did not induce apoptosis on their own nor did they enhance the apoptotic response in a model of Ca2+-dependent, NCX-independent apoptosis. Additional control experiments demonstrated that the NCX antisense oligonucleotide did not alter the apoptotic response of myocytes to either H2O2 or isoproterenol. Taken together, these data suggest that the NCX has a critical and specific role in the initiation of apoptosis after hypoxia-reoxygenation in guinea pig myocytes and that hypoxia-reoxygenation-induced apoptosis is quite sensitive to changes in NCX activity.

Leukotriene B4(LTB4) is a potent leukocyte chemoattractant recently implicated in the pathogenesis of atherosclerosis. The aim of this study was to assess the effects of LTB4on isolated aortic preparations. Rings of guinea pig aorta were challenged with LTB4for recording mechanical responses and measurements of mediator release, and LTB4receptor (BLT1) expression was assessed by RT-PCR. Single concentrations of LTB4induced concentration-dependent contractions that were inhibited by treatment with antihistamines, indomethacin, or the thromboxane receptor antagonist BAYu3405 as well as by denudation of endothelium. In addition, LTB4increased the release of histamine and thromboxane in the bath. The contractions induced by LTB4were inhibited by either the unselective BLT receptor antagonist ONO-4057 or the selective BLT1receptor antagonist U-75302. Pretreatment with all -trans-retinoic acid enhanced the contractions and the release of histamine induced by LTB4, without affecting either the contractions induced by histamine or the histamine release evoked by calcium ionophore A23187. Analysis by RT-PCR indicated the expression of a BLT1receptor in the guinea pig aorta and that BLT1receptor mRNA was upregulated after treatment with retinoic acid. These results suggest that LTB4contracts the guinea pig aorta via an indirect mechanism involving the release of histamine and thromboxane and that this BLT1receptor-mediated response can be upregulated by all -trans-retinoic acid.

Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200–600 nM) evoked depolarizing responses (8.2 ± 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.

Activation of cardiac mast cells has been shown to alter parasympathetic neuronal function via the activation of histamine receptors. The present study examined the ability of prostaglandins to alter the activity of guinea pig intracardiac neurons. Intracellular voltage recordings from whole mounts of the cardiac plexus showed that antigen-mediated mast cell degranulation produces an attenuation of the afterhyperpolarization (AHP), which was prevented by the phospholipase A2 inhibitor 5,8,11,14-eicosatetraynoic acid. Exogenous application of either PGD2 or PGE2 produced a biphasic change in the membrane potential and an inhibition of both AHP amplitude and duration. Examination of prostanoid receptors using bath perfusions (1 μM PGE2 and PGD2), specific agonists (BW245C, sulprostone, and butaprost), and antagonists (AH6809 and SC19220) found evidence for both the PGE2-specific EP2 and EP3 receptors, but not for EP1 or the PGD2-specific prostanoid (DP) receptors. Sulprostone was able to mimic the PGE2 responses in some cells, but not in all PGE2-sensitive cells. Butaprost was able to mimic the PG-induced hyperpolarization in some cells, but did not alter the AHP. Inhibition of specific potassium channels with either TEA, charybdotoxin, or apamin showed that neither TEA nor charybdotoxin could prevent the PGE2-induced AHP attenuation. Apamin alone inhibited AHP duration, with PGs having no further effect in these cells. These results demonstrate that guinea pig intracardiac neurons can be modulated by PG, most likely through either EP2, EP3, or potentially EP4 receptors, and this response is due, at least in part, to a reduction in small-conductance KCa currents.

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E2(PGE2) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 ± 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 ± 1.8 and 14.3 ± 1.7% compared with K+(72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP1antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE2induced only a slight phasic contraction in guinea pig trachea, but PGE2induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE2generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE2production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP1receptor.

Primary cultures of guinea pig gastric mucosal cells express NADPH oxidase 1 (Nox1), a homolog of gp91phox, and produce superoxide anion (O2−) at a rate of ∼100 nmol·mg protein−1·h−1in response to Helicobacter pylori ( H. pylori) lipopolysaccharide (LPS) from virulent type I strains. The upregulated O2−production also enhances H. pylori LPS-stimulated tumor necrosis factor-α or cyclooxygenase-2 mRNA expression, which suggests a potential role for Nox1 in the pathogenesis of H. pylori-associated diseases. The H. pylori LPS-stimulated O2−production in cultured gastric mucosal cells was inhibited by actinomycin D as well as cycloheximide, suggesting that the induction is regulated at the transcriptional level. The LPS treatment not only increased the Nox1 mRNA to a greater extent but also induced expression of the message-encoding, Nox-organizing protein 1 (NOXO1), a novel p47phoxhomolog required for Nox1 activity. In addition, H. pylori LPS activated Rac1; i.e., it converted Rac1 to the GTP-bound state. A phosphoinositide 3-kinase inhibitor, LY-294002, blocked H. pylori LPS-induced Rac1 activation and O2−generation without interfering with the expression of Nox1 and NOXO1 mRNA. O2−production inhibited by LY-294002 was completely restored by transfection of an adenoviral vector encoding a constitutively active Rac1 but not an inactive Rac1 or a constitutively active Cdc42. These findings indicate that Rac1 plays a crucial role in Nox1 activation. Thus the H. pylori LPS-stimulated O2−production in gastric mucosal cells appears to require two distinct events: 1) transcriptional upregulation of Nox1 and NOXO1 and 2) activation of Rac1.

The motility of the gut depends on the chemicals contained in the lumen, but the stimuli that modify motility and their relationship to enteric neural pathways are unclear. This study examined local inhibitory reflexes activated by various chemical stimulants applied to the mucosa to characterize effective physiological stimuli and the pathways they excite. Segments of the jejunum were dissected to allow access to the circular muscle on one-half of the preparation while leaving the mucosa intact on the circumferentially adjacent half. Chemicals were transiently applied to the mucosa, and responses were recorded intracellularly in nearby circular muscle cells. The amino acids l-phenylalanine, l-alanine, or l-tryptophan (all 1 mM) evoked inhibitory junction potentials (IJPs; latency 150–300 ms, amplitude 3–8 mV, each n > 6) that were blocked by TTX and partially blocked by antagonists of P2X receptors and/or a combination of antagonists at 5-HT3 and 5-HT4 receptors. The putative mediators 5-HT (10 μM), ATP (1 mM), and CCK-8 (1–10 μM) elicited IJPs mediated via 5-HT3, P2X, and CCK-B receptors, respectively. Responses were only partially reduced by the effective antagonists. IJPs evoked by electrically stimulating the mucosa were unaffected by antagonists that reduced chemically evoked responses. Both chemically and electrically evoked IJPs were resistant to nicotinic, NK1, NK3, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-d-aspartate, or CGRP receptor blockade. We conclude that mucosal stimulation by amino acids activates local neural pathways whose pharmacology depends on the nature of the stimulus. Transmitters involved at some synapses in these pathways remain to be identified.

Extracellular recordings were obtained from single cochlear nucleus neurons in guinea pigs anesthetized with Nembutal and Hypnorm. Neurons were classified by their spontaneous firing rates and responses to acoustic stimuli. In addition, electrical shocks were applied to the midline at the level of the IVth ventricle and spike responses were recorded. Spikes were evoked by shocks only in neurons that were classified as onset choppers (Oc). The shock-evoked spikes could be extinguished by acoustically evoked action potentials in the same neurons. In roughly 30% of the sample of Oc neurons, quantitative aspects of the timing of this extinction were not compatible with the shock-evoked spike being antidromically conducted from Oc output axons. Together with the presence of temporal jitter at high shock rates, the data suggest the possibility that at least some of the shock-evoked spikes may be generated by excitatory synaptic input to the Oc neurons, most likely from the collaterals of the medial olivocochlear system (MOCS), whose axons pass close to the floor of the IVth ventricle. This excitatory synaptic input may operate to modulate the activity of Oc neurons in addition to MOCS actions in the auditory periphery.

Ca+ waves have been implicated in Ca2+ overload-induced cardiac arrhythmias. To deepen understanding of the behavior of Ca2+ waves in a multicellular system, consecutive two-dimensional Ca2+ images were obtained with a confocal microscope from surface cells of guinea pig ventricular papillary muscles loaded with fluo 3 or rhod 2. In intact muscles, no Ca2+ waves were detected under the resting condition, whereas they were frequently observed during the rest immediately after high-frequency stimulations where cytoplasmic Ca2+ concentration and Ca2+ stored in the sarcoplasmic reticulum (SR) were gradually decreasing. The intervals of Ca2+ waves increased as they occurred later, their amplitudes and velocities remaining unchanged. A SERCA inhibitor reversibly prolonged the wave intervals. In Na+-free/Ca2+-free medium where neither Ca2+ influx nor Na+/Ca2+ exchange took place, recurrent Ca2+ waves emerged at constant intervals in each cell. These results are consistent with the conclusion that the loading level of the SR is critical for induction of Ca2+ waves. Each cell independently exhibited its own regular rhythm of Ca2+ wave with a distinct interval. These waves propagated in either direction along the longitudinal axis within a muscle cell, but seldom beyond the cell boundary. In contrast, in partially damaged muscles that showed spontaneous Ca2+ waves at rest in normal Krebs solution, their propagation often was unidirectional, decreasing in frequency. In these cases, however, Ca2+ waves rarely moved beyond the cellular boundary. The gradient of the cytoplasmic Ca2+ concentration was suggested to be the cause of the one-way propagation.

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+current ( ICa,L) in myocytes dialyzed with 10 mM K+solution and superfused with Tyrode’s solution. Although depolarizations from holding potential ( Vhp) −40 to 0 mV elicited relatively small inward ICa,Lin these myocytes, removal of external K+or addition of 0.2 mM Ba2+more than doubled the amplitude of the current. The basis of the enhancement of ICa,Lwas the suppression of a large transient outward K+current. Similar enhancement was observed when Vhpwas moved to −80 mV and test depolarizations were preceded by short prepulses to −40 mV. Investigation of the time and voltage properties of the outward K+transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+solution, accelerated in myocytes dialyzed with 100 μM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic “time-independent” inwardly rectifying K+current.

Currents carried by L-, N-, and P/Q-type calcium channels do not account for the total calcium current in myenteric neurons. This study identified all calcium channels expressed by guinea pig small intestinal myenteric neurons maintained in primary culture. Calcium currents were recorded using whole cell techniques. Depolarizations (holding potential = −70 mV) elicited inward currents that were blocked by CdCl2 (100 μM). Combined application of nifedipine (blocks L-type channels), Ω-conotoxin GVIA (blocks N-type channels), and Ω-agatoxin IVA (blocks P/Q-type channels) inhibited calcium currents by 56%. Subsequent addition of the R-type calcium channel antagonists, NiCl2 (50 μM) or SNX-482 (0.1 μM), abolished the residual calcium current. NiCl2 or SNX-482 alone inhibited calcium currents by 46%. The activation threshold for R-type calcium currents was −30 mV, the half-activation voltage was −5.2 ± 5 mV, and the voltage sensitivity was 17 ± 3 mV. R-type currents activated fully in 10 ms at 10 mV. R-type calcium currents inactivated in 1 s at 10 mV, and they inactivated (voltage sensitivity of 16 ± 1 mV) with a half-inactivation voltage of −76 ± 5 mV. These studies have accounted for all of the calcium channels in myenteric neurons. The data indicate that R-type calcium channels make the largest contribution to the total calcium current in myenteric neurons. The relatively positive half-activation voltage and rapid activation kinetics suggest that R-type channels could contribute to calcium entry during somal action potentials or during action potential-induced neurotransmitter release.

Guinea pig mesenteric lymphatic vessels exhibit vasomotion through a pacemaker mechanism that involves intracellular Ca2+release and resultant spontaneous transient depolarizations (STDs) of the smooth muscle membrane potential. This study presents a detailed characterization of the effects of cyclopiazonic acid (CPA) on this pacemaker activity. Microelectrode recordings from smooth muscle in vessel segments revealed that application of CPA (1–10 μM) caused a hyperpolarization accompanied by a decrease in the frequency and amplitude of STDs. The CPA-induced hyperpolarization was abolished after destruction of the endothelium and in the presence of NG-nitro-l-arginine (100 μM) or 1 H-[1,2,4]oxadiazolol-[4,3- a]quinoxaline-1-one (10 μM), which suggests a contribution of endothelium-derived nitric oxide (EDNO) in this response. In the absence of EDNO-induced effects, CPA decreased the frequency and amplitude of STDs recorded before and in the presence of the thromboxane A2mimetic U-46619, norepinephrine, or thimerosal. CPA abolished U-46619-induced vasomotion as determined by measurement of constriction-associated intracellular Ca2+concentration using the ratiometric Ca2+indicator fura-2. The endothelial actions of CPA were compared with those of ACh, which is known to cause EDNO release in this preparation. Although CPA and ACh both increased endothelial intracellular Ca2+concentration and depolarized the membrane potential, the kinetics of action for both parameters were markedly slower for CPA than ACh. These results suggest that CPA first hyperpolarizes the lymphatic smooth muscle and decreases STD frequency and amplitude through endothelial release of EDNO, and second, consistent with the action of CPA to inhibit sarcoplasmic reticulum Ca2+-ATPase and deplete Ca2+stores, it further reduces STD activity. Inhibition of the lymphatic smooth muscle pacemaker mechanism is thought to abolish agonist-induced vasomotion.

—Application of substance P (SP) to intracardiac neurons of the guinea pig causes slow depolarization and increases neuronal excitability. The present study was done to determine the influence of SP on fast excitatory responses of intracardiac neurons to ACh. Intracellular recording methods were used to measure responses of intracardiac neurons in whole mount preparations of atrial ganglionated nerve plexus from guinea pig hearts. Local pressure ejection of 100 μM SP (1 s) from a glass micropipette caused slow depolarization of all neurons ( n = 38) and triggered action potential generation in 47% of the cells tested. Bath application of SP (0.5–100 μM) caused a dose-dependent depolarization of intracardiac neurons but rarely evoked action potentials, even at the highest concentration. However, such treatment with SP enhanced nicotinic responses evoked by local pressure ejections of ACh (10 mM, 10- to 100-ms duration) in 77% of intracardiac neurons studied ( n = 52). A significant increase in amplitude of ACh-evoked fast depolarization occurred during treatment with 0.5 μM SP (13.0 ± 1.8 mV for control vs. 17.7 ± 1.9 mV with SP present, n = 7, P = 0.019). At higher concentrations of SP, enhancement of the response to ACh resulted mainly in action potential generation. However, responses to ACh were attenuated by SP in 15% of the intracardiac neurons studied. This attenuation occurred primarily during exposure to 10 and 100 μM SP and was manifest as a reduction in amplitude of nicotinic fast depolarization or inhibition of ACh-evoked action potentials. These findings support the conclusion that SP could function as a neuromodulator and neurotransmitter in intracardiac ganglia of the guinea pig.

The Gibbs free energy of the sarcolemmal Na+/Ca2+exchanger (ΔGNa/Ca) determines its net Ca2+flux. We tested the hypothesis that a difference of diastolic ΔGNa/Caexists between rat and guinea pig myocardium. We measured the suprabasal rate of oxygen consumption (Vo2) of arrested Langendorff-perfused hearts of both species, manipulating ΔGNa/Caby reduction of extracellular Na+concentration, [Na+]o. Hill equations fitted to the resulting Vo2-[Na+]orelationships yielded Michaelis constant ( Km) values of 67 and 25 mM for rat and guinea pig, respectively. We developed and tested a simple thermodynamic model that attributes this difference of Kmvalues to a 7.84 kJ/mol difference of ΔGNa/Ca. The model predicts that reversal of Na+/Ca2+exchange, leading to diastolic Ca2+influx, should occur at a value of [Na+]oabout three times higher in rat myocardium. We verified this quantitative prediction using fura 2 fluorescence to index intracellular Ca2+concentration in isolated ventricular trabeculae at 37°C. The postulated difference in free energy of Na+/Ca2+exchange explains a number of reported disparities of Ca2+handling at rest between rat and guinea pig myocardia.

The effect of denopamine, a selective β1-adrenergic agonist, on alveolar fluid clearance was determined in both ex vivo rat and guinea pig lungs. Alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue-labeled albumin over 1 h at 37°C. Denopamine (10−6 to 10−3 M) increased alveolar fluid clearance in a dose-dependent manner in ex vivo rat lungs. Denopamine also stimulated alveolar fluid clearance in guinea pig lungs. Atenolol, a selective β1-adrenergic antagonist, and amiloride, a sodium channel inhibitor, inhibited denopamine-stimulated alveolar fluid clearance. The potency of denopamine was similar to that of similar doses of isoproterenol or terbutaline. Short-term hypoxia (100% nitrogen for 1–2 h) did not alter the stimulatory effect of denopamine. Denopamine (10−4, 10−3 M) increased intracellular adenosine 3′,5′-cyclic monophosphate levels in cultured rat alveolar type II cells. In summary, denopamine, a selective β1-adrenergic agonist, stimulates alveolar fluid clearance in both ex vivo rat and guinea pig lungs.

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 10–4 M), the neuronal blocker tetrodotoxin (TTX, 10–6 M), or both on spontaneous contractions and on the production of basal NO. Both l-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by l-NAME and was inhibited by 38% by TTX. When tested together, l-NAME in the presence of TTX or TTX in the presence of l-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.

Cell-attached recordings revealed Cl− channel activity in basolateral membrane of guinea pig distal colonic crypts isolated from basement membrane. Outwardly rectified currents (gpClor) were apparent with a single-channel conductance (γ) of 29 pS at resting membrane electrical potential; another outward rectifier with γ of 24 pS was also observed (∼25% of gpClor). At a holding potential of −80 mV γ was 18 pS for bothgpClor currents, and at +80 mV γ was 67 and 40 pS, respectively. Identity as Cl− channels was confirmed in excised patches by changing bath ion composition. From reversal potentials, relative permeability of K+ over Cl− ( P K/ P Cl) was 0.07 ± 0.03, with relative permeability of Na+over Cl−( P Na/ P Cl) = 0.08 ± 0.04. A second type of Cl− channel was seen with linear current-voltage ( I-V) relations (gpClL), having subtypes with γ of 21, 13, and 8 pS. Epinephrine or forskolin increased the number of opengpClor and gpClL. Open probabilities ( P o) ofgpClor, gpClL21, andgpClL13 were voltage dependent in cell-attached patches, higher at more positive potentials. Kinetics ofgpClor were more rapid with epinephrine activation than with forskolin activation. Epinephrine increased P o at the resting membrane potential forgpClL13. Secretagogue activation of these Cl− channels may contribute to stimulation of electrogenic K+ secretion across colonic epithelium by increasing basolateral membrane Cl− conductance that permits Cl− exit after uptake via Na+-K+-2Cl− cotransport.

The effect of reducing extracellular calcium concentration ([Ca2+]o) on vagal afferent excitability was analyzed in a guinea pig isolated vagally innervated trachea-bronchus preparation. Afferent fibers were characterized as either having low-threshold, rapidly adapting mechanosensors (Aδ fibers) or nociceptive-like phenotypes (Aδ and C fibers). The nociceptors were derived from neurons within the jugular ganglia, whereas the low-threshold mechanosensors were derived from neurons within the nodose ganglia. Reducing [Ca2+]o did not affect the excitability of the low-threshold mechanosensors in the airway. By contrast, reducing [Ca2+]o selectively increased the excitability of airway nociceptors as manifested by a substantive increase in action potential discharge in response to mechanical stimulation, and in a subset of fibers, by overtly evoking action potential discharge. This increase in the excitability of nociceptors was not mimicked by a combination of ω-conotoxin and nifedipine or tetraethylammonium. Whole cell patch recordings from airway-labeled and unlabeled neurons in the vagal jugular ganglia support the hypothesis that [Ca2+]o inhibits a nonselective cation conductance in vagal nociceptors that may serve to regulate excitability of the nerve terminals within the airways.

Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.

A novel transient outward K+current that exhibits inward-going rectification ( I to.ir) was identified in guinea pig atrial and ventricular myocytes. I to.ir was insensitive to 4-aminopyridine (4-AP) but was blocked by 200 μmol/l Ba2+or removal of external K+. The zero current potential shifted 51–53 mV/decade change in external K+. I to.ir density was twofold greater in ventricular than in atrial myocytes, and biexponential inactivation occurs in both types of myocytes. At −20 mV, the fast inactivation time constants were 7.7 ± 1.8 and 6.1 ± 1.2 ms and the slow inactivation time constants were 85.1 ± 14.8 and 77.3 ± 10.4 ms in ventricular and atrial cells, respectively. The midpoints for steady-state inactivation were −36.4 ± 0.3 and −51.6 ± 0.4 mV, and recovery from inactivation was rapid near the resting potential (time constants = 7.9 ± 1.9 and 8.8 ± 2.1 ms, respectively). I to.ir was detected in Na+-containing and Na+-free solutions and was not blocked by 20 nmol/l saxitoxin. Action potential clamp revealed that I to.ir contributed an outward current that activated rapidly on depolarization and inactivated by early phase 2 in both tissues. Although it is well known that 4-AP-sensitive transient outward current is absent in guinea pig, this Ba2+-sensitive and 4-AP-insensitive K+ current has been overlooked.

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of −50 ± 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca2+channels with nifedipine or by depleting intracellular Ca2+stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 μM). In quiescent cells, nifedipine (1 μM) reduced EJPs by 30%, whereas ryanodine (10 μM) reduced EJPs by 93%. These results suggest that both the release of Ca2+ from intracellular stores and the influx of Ca2+ through voltage-dependent Ca2+channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.

Previous studies have documented that repetitive exposure to intermittent hypoxia, such as that encountered in preparation to high-altitude ascent, influences breathing. However, the impact of intermittent hypoxia on airway smooth muscle has not been explored. Ascents to high altitude, in addition to hypoxia, expose individuals to cold air. The objective of the present study is to examine the effect of chronic intermittent hypobaric hypoxia (CIH) and CIH combined with cold exposure (CIHC) on tracheal smooth muscle responses to various contractile and relaxant agonists. Experiments were performed on tracheal rings harvested from adult guinea pigs exposed either to CIH or CIHC [14 days (6 h/day) at barometric pressure of 350 mmHg with and without cold exposure of 5°C] or to room air (normoxia). CIH and CIHC attenuated maximum contractile responses to ACh compared with normoxia. The maximum contractile response to histamine decreased with CIH, whereas CIHC restored the response back to normoxia. Both CIH and CIHC attenuated maximum contractile responses to 5-HT. Altered contractile responses after CIH and CIHC were independent of epithelium. Isoproterenol-induced relaxation was not altered by CIH, whereas it was enhanced after CIHC, and these responses were independent of the epithelium. The data demonstrate that intermittent exposure to hypoxia profoundly influences contractile response of tracheal smooth muscle, and cold exposure can further modulate the response, implying the importance of cold at high altitude.

Previous work from this laboratory using near-infrared optical spectroscopy of myoglobin has shown that approximately 20% of the myocardium is hypoxic in buffer-perfused hearts that are perfused with fully oxygenated buffer at 37 degrees C. The present study was undertaken to determine cardiac myoglobin saturation in buffer-perfused hearts when cardiac contractility was increased with epinephrine and decreased during cardiac arrest with KCl. Infusion of epinephrine to achieve a doubling of contractility, as measured by left ventricular maximum pressure change over time (dP/dt), resulted in a decrease in mean myoglobin saturation from 79% at baseline to 65% and a decrease in coronary venous oxygen tension from 155 mmHg at baseline to 85 mmHg. Cardiac arrest with KCl increased mean myoglobin saturation to 100% and coronary venous oxygen tension to 390 mmHg. A previously developed computer model of oxygen transport in the myocardium was used to calculate the probability distribution of intracellular oxygen tension and the hypoxic fraction of the myocardium with an oxygen tension below 0.5 mmHg. The hypoxic fraction of the myocardium was approximately 15% at baseline, increased to approximately 30% during epinephrine infusion, and fell to approximately 0% during cardiac arrest. The coronary venous adenosine concentration changed in parallel with the hypoxic fraction of the myocardium during epinephrine and KCl. It is concluded that catecholamine stimulation of buffer-perfused hearts increases hypoxia in the myocardium and that the increase in venous adenosine concentration is a reflection of the larger hypoxic fraction of myocardium that is releasing adenosine.

The aim of this study was to delineate the mode of action of 20-hydroxy-eicosatetraenoic acid (20-HETE) in airway smooth muscle (ASM) cells. ASM metabolizes arachidonic acid by various enzymatic pathways, including the cytochrome P-450 (CYP-450) ω-hydroxylase, which leads to the production of 20-HETE, a bronchoconstrictive eicosanoid. The present study demonstrated that 20-HETE induced concentration-dependent tonic responses in ASM, whereas transient responses were recorded in Ca2+-free solution, suggesting an intracellular Ca2+release process. 20-HETE inotropic responses were abolished by 36 μM 2-aminoethoxydiphenyl borate or 1 μM thapsigargin but were insensitive to 10 μM ryanodine, indicating that inositol triphosphate receptors likely control the release of intracellular Ca2+. Sustained tension, which required Ca2+entry, was partially blocked by 1 μM nifedipine (an L-type) and 100 μM Gd3+(a nonselective cationic channel blocker). Moreover, in the absence of selective 20-HETE receptor antagonists, 20-HETE tonic responses were inhibited in a concentration-dependent manner (0.1–10 μM) by capsazepine, a well-characterized vanilloid receptor antagonist. Capsazepine was also observed to reverse cumulative responses to 20-HETE and capsaicin, a TRPV1 agonist. In addition, capsazepine pretreatment largely modified the sustained inotropic responses to 20-HETE, suggesting that 20-HETE cross-reacted with TRPV1 receptors with a low affinity (μM) or that its specific receptor was inhibited by the vanilloid antagonist. Data obtained using RHC-80267, ONO-RS-082, and eicosatetraynoic acid, respective inhibitors of diacylglycerol-lipase, phospholipase A2, and CYP-450 ω-hydroxylase, reveal that intracellular arachidonic acid production and its 20-HETE metabolite may be responsible for the activation of nonselective cationic channels and tonic responses.

Neurokinin-containing nerve fibers were localized to guinea pig airway parasympathetic ganglia in control tissues but not in tissues pretreated with capsaicin. The purpose of the present study was to determine whether neurokinins, released during axonal reflexes or after antidromic afferent nerve stimulation, modulate ganglionic synaptic neurotransmission. The neurokinin type 3 (NK3) receptor antagonists SB-223412 and SR-142801 inhibited vagally mediated cholinergic contractions of bronchi in vitro at stimulation voltages threshold for preganglionic nerve activation but had no effect on vagally mediated contractions evoked at optimal voltage or field stimulation-induced contractions. Intracellular recordings from the ganglia neurons revealed that capsaicin-sensitive nerve stimulation potentiated subsequent preganglionic nerve-evoked fast excitatory postsynaptic potentials. This effect was mimicked by the NK3 receptor agonist senktide analog and blocked by SB-223412. In situ, senktide analog markedly increased baseline tracheal cholinergic tone, an effect that was reversed by atropine and prevented by vagotomy or SB-223412. Comparable effects of intravenous senktide analog on pulmonary insufflation pressure were observed. These data highlight the important integrative role played by parasympathetic ganglia and indicate that activation of NK3 receptors in airway ganglia by endogenous neurokinins facilitates synaptic neurotransmission.

In the vagal-sensory system, neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) are synthesized nearly exclusively in small-diameter nociceptive type C-fiber neurons. By definition, these neurons are designed to respond to noxious or tissue-damaging stimuli. A common feature of visceral inflammation is the elevation in production of sensory neuropeptides. Little is known, however, about the physiological characteristics of vagal sensory neurons induced by inflammation to produce substance P. In the present study, we show that allergic inflammation of guinea pig airways leads to the induction of substance P and CGRP production in large-diameter vagal sensory neurons. Electrophysiological and anatomical evidence reveals that the peripheral terminals of these neurons are low-threshold Aδ mechanosensors that are insensitive to nociceptive stimuli such as capsaicin and bradykinin. Thus inflammation causes a qualitative change in chemical coding of vagal primary afferent neurons. The results support the hypothesis that during an inflammatory reaction, sensory neuropeptide release from primary afferent nerve endings in the periphery and central nervous system does not require noxious or nociceptive stimuli but may also occur simply as a result of stimulation of low-threshold mechanosensors. This may contribute to the heightened reflex physiology and pain that often accompany inflammatory diseases.

Cell-attached recordings revealed K+ channel activity in basolateral membranes of guinea pig distal colonic crypts. Inwardly rectified currents were apparent with a pipette solution containing 140 mM K+. Single-channel conductance (γ) was 9 pS at the resting membrane potential. Another inward rectifier with γ of 19 pS was observed occasionally. At a holding potential of −80 mV, γ was 21 and 41 pS, respectively. Identity as K+ channels was confirmed after patch excision by changing the bath ion composition. From reversal potentials, relative permeability of Na+ over K+ ( P Na/ P K) was 0.02 ± 0.02, with P Rb/ P K = 1.1 and P Cl/ P K < 0.03. Spontaneous open probability ( P o) of the 9-pS inward rectifier (gpKir) was voltage independent in cell-attached patches. Both a low ( P o = 0.09 ± 0.01) and a moderate ( P o = 0.41 ± 0.01) activity mode were observed. Excision moved gpKir to the medium activity mode; P o ofgpKir was independent of bath Ca2+activity and bath acidification. Addition of Cl− and K+ secretagogues altered P o ofgpKir. Forskolin or carbachol (10 μM) activated the small-conductance gpKir in quiescent patches and increased P o in low-activity patches. K+ secretagogues, either epinephrine (5 μM) or prostaglandin E2 (100 nM), decreased P o of gpKir in active patches. This gpKir may be involved in electrogenic secretion of Cl− and K+ across the colonic epithelium, which requires a large basolateral membrane K+ conductance during maximal Cl− secretion and, presumably, a lower K+ conductance during primary electrogenic K+ secretion.

Short-term stimulation of β-receptors is known to affect cardiac ion channels; however, the impact of longer-term stimulation on intrinsic channel function is poorly understood. To evaluate this, cultured guinea pig ventricular myocytes were exposed to isoproterenol (10 nM), vehicle, or isoproterenol plus propranolol (1 μM) for 48 h. Sustained exposure to isoproterenol decreased the density of the inward rectifier ( I K1), slow delayed rectifier ( I Ks), and L-type Ca2+( I Ca L) currents, effects that were fully prevented by propranolol. Changes in K+ currents were prevented by the β1-selective antagonist CGP-20712A, unaffected by the β2-antagonist ICI-118,551, and mimicked by the membrane-permeable cAMP analog 8-bromo-cAMP. Isoproterenol did not alter the current-voltage relationship of the K+currents but increased the density of T-type Ca2+ current ( I Ca T) and thereby increased the proportion of the total Ca2+ current at more negative potentials. We conclude that sustained exposure to isoproterenol reduces I K1, I Ks, and I Ca L density and increases the density of I Ca T. The direct ionic current remodeling effects of sustained β-adrenoceptor stimulation resemble changes reported with heart failure and may be important in arrhythmogenic ionic remodeling.