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Pituitary adenylate cyclase- activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/ PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Cav3.1, Cav3.2, and Cav3.3 T-type isoforms and R-type Cav2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization- induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents (Ih). The T-type calcium channel inhibitors, nickel (Ni2+) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2-4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni2+, a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAPinitiated increase in excitability. Ni2+ also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni2+-sensitive current contributes to PACAP-induced modulation of neuronal excitability. [ABSTRACT FROM AUTHOR]

The elevation of protein kinase A (PKA) activity activates the largeconductance voltage- and Ca2+-activated K+ (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca2+ sparks but increased global Ca2+ levels and the magnitude of Ca2+ oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca2+ signals generated by intracellular Ca2+ stores and cytosolic Ca2+ levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM. [ABSTRACT FROM AUTHOR]

The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca2+ channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATPbiotin maintains Ca2+ channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dosedependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca2+ was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509 -1,789) of the main subunit (ɑ1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca2+ dependent. [ABSTRACT FROM AUTHOR]

Mechanisms underlying ethanol (EtOH)-induced detrusor smooth muscle (DSM) relaxation and increased urinary bladder capacity remain unknown. We investigated whether the large conductance Ca2+-activated K+ (BK) channels or L-type voltage-dependent Ca channels (VDCCs), major regulators of DSM excitability and contractility, are targets for EtOH by patch-clamp electrophysiology (conventional and perforated whole cell and excised patch single channel) and isometric tension recordings using guinea pig DSM cells and isolated tissue strips, respectively. EtOH at 0.3% vol/vol (50 mM) enhanced whole cell BK currents at30 mV and above, determined by the selective BK channel blocker paxilline. In excised patches recorded at +40 mV and ~300 nM intracellular Ca2+ concentration ([Ca2+]), EtOH (0.1-0.3%) affected single BK channels (mean conductance ~210 pS and blocked by paxilline) by increasing the open channel probability, number of open channel events, and open dwell-time constants. The amplitude of single BK channel currents and unitary conductance were not altered by EtOH. Conversely, at ~10 µM but not ~2 µM intracellular [Ca2+], EtOH (0.3%) decreased the single BK channel activity. EtOH (0.3%) affected transient BK currents (TBKCs) by either increasing frequency or decreasing amplitude, depending on the basal level of TBKC frequency. In isolated DSM strips, EtOH (0.1-1%) reduced the amplitude and muscle force of spontaneous phasic contractions. The EtOH-induced DSM relaxation, except at 1%, was attenuated by paxilline. EtOH (1%) inhibited L-type VDCC currents in DSM cells. In summary, we reveal the involvement of BK channels and L-type VDCCs in mediating EtOH-induced urinary bladder relaxation accommodating alcohol-induced diuresis. [ABSTRACT FROM AUTHOR]

Members of the transient receptor potential (TRP) channel superfamily, including the Ca2+-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca2+ imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca2+ imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca2+ levels. 9-Phenanthrol (0.1-30 μM) significantly inhibited spontaneous, 0.1 μM carbacholinduced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1-7 μM and 70-80% maximum inhibition. 9-Phenanthrol also reduced nerveevoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequencyresponse curve (0.5-50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling. [ABSTRACT FROM AUTHOR]

The secretin-stimulated human pancreatic duct secretes HCO3--rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO3- secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl-/HCO3- exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO3- or more, mouse and rat ducts secrete ∼40-70 mM HCO3-. Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO3- secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl-/Cl- exchange and electroneutral Cl-/HCO3- exchange. gpSlc26a6 in Xenopus oocytes mediated Cl-/Cl- exchange and bidirectional exchange of Cl- for oxalate and sulfate, but Cl-/HCO3- exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl-, oxalate, and sulfate transport but no detectable Cl-/HCO3- exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of 36Cl- influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO3-secretion in species that share a high HCO3- secretory output. [ABSTRACT FROM AUTHOR]

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 μM or higher suppressed Ca2+-induced tension development at any given Ca2+ concentration but had little effects on the Ca2+-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.75 mM Mg2+-induced, "myosin light chain phosphorylation-independent" tension development at more than 10 μM. 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 Ca2+ In addition, blebbistatin partially inhibited Mg2+- ATPase activity of native actomyosin from guinea pig taenia cecum at around 10 μM. These results suggested that blebbistatin suppressed skinned smooth muscle contraction through disruption of structure of SMM by the agent. [ABSTRACT FROM AUTHOR]

Large-conductance Ca2+-activated K+ channels (BK+a) in the inner mitochondrial membrane may play a role in protecting against cardiac ischemia-reperfusion injury. NS1619 (30 μM), an activator of BKca channels, was shown to increase respiration and to stimulate reactive oxygen species generation in isolated cardiac mitochondria energized with succinate. Here, we tested effects of NS1619 to alter matrix K+, H+, and swelling in mitochondria isolated from guinea pig hearts. We found that 30 μM NS1619 did not change matrix K+, Ht and swelling, but that 50 and 100 μM NSI619 caused a concentrationdependent increase in matrix K+ influx (PBFI fluorescence) only when quinine was present to block K+/H+ exchange (KHE); this was accompanied by increased mitochondrial matrix volume (light scattering). Matrix pH (BCECF fluorescence) was decreased slightly by 50 and 100 pM NS1619 but markedly more so when quinine was present. NS1619 (100 μM) caused a significant leak in lipid bilayers, and this was enhanced in the presence of quinine. The K+ ionophore valinomycin (0.25 nM), which like NS1619 increased matrix volume and increased K+ influx in the presence of quinine, caused matrix alkalinization followed by acidification when quinine was absent, and only alkalinization when quinine was present. If K+ is exchanged instantly by H+ through activated KHE, then matrix K+ influx should stimulate H+ influx through KHE and cause matrix acidification. Our results indicate that KHE is not activated immediately by NS1619 induced K+ influx, that NS1619 induces matrix K+ and H+ influx through a nonspecific transport mechanism, and that enhancement with quinine is not due to the blocking of KHE, but to a nonspecific effect of quinine to enhance current leak by NS1619. [ABSTRACT FROM AUTHOR]

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the Ca2+-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the Ca2+-dependent force development was partially restored, but the Ca2+-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its Ca2+ sensitivity and the reversible Ca2+-dependent reduction in CS4 We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and Carp are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin- linked one. [ABSTRACT FROM AUTHOR]

Components of excitation- contraction (EC)-coupling were compared at 37°C and 22°C to determine whether hypothermia altered the gain of EC coupling in guinea pig ventricular myocytes. Ca2+ concentration (fura-2) and cell shortening (edge detector) were measured simultaneously. Hypothermia increased fractional shortening (8.3 ± 1.7 vs. 2.6 ± 0.3% at 37°C), Ca2+ transients (157 ± 33 vs. 35 ± 5 nM at 37°C), and diastolic Ca2+ (100 ± 9 vs. 60 ± 6 nM at 37°C) in field-stimulated myocytes (2 Hz). In experiments with high-resistance microelectrodes, the increase in contractions and Ca2+ transients was accompanied by a twofold increase in action potential duration (APD). When voltage-clamp steps eliminated changes in APD, cooling still increased contractions and Ca2+ transients. Hypothermia increased sarcoplasmic reticulum (SR) Ca2+ stores (83 ± 17 at 37°C to 212 ± 50 nM, assessed with caffeine) and increased fractional SR Ca2+ release twofold. In contrast, peak Ca2+ current was much smaller at 22°C than at 37°C (1.3 ± 0.4 and 3.5 ± 0.7 pA/pF, respectively). In cells dialyzed with sodium-free pipette solutions to inhibit Ca2+ influx via reverse-mode Na+/Ca2+ exchange, hypothermia still increased contractions, Ca2+ transients, SR stores, and fractional release but decreased the amplitude of Ca2+ current. The rate of SR Ca2+ release per unit Ca2+ current, a measure of EC-coupling gain, was increased sixfold by hypothermia. This increase in gain occurred regardless of whether cells were dialyzed with sodium-free solutions. Thus an increase in EC-coupling gain contributes importantly to positive inotropic effects of hypothermia in the heart. [ABSTRACT FROM AUTHOR]

The inhibitory control of pancreatic ductal HCO3- secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO3- secretion by modulating a Cl- -dependent HCO3- efflux step at the apical membrane of the duct cell (Hegyi P, Gray MA, and Argent BE. Am J Physiol Cell Physiol 285: C268-C276, 2003). In the present study, we have shown that SP is present in periductal nerves within the guinea pig pancreas, that PKC mediates the effect of SP, and that SP inhibits an anion exchanger on the luminal membrane of the duct cell. Secretin (10 nM) stimulated HCO3- secretion by sealed, nonperfused, ducts about threefold, and this effect was totally inhibited by SP (20 nM). Phorbol 12,13.-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO3- secretion by ∼40% and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1 µM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO3- secretion and also reversed the inhibitory effect of PDBu. Western blot analysis revealed that guinea pig pancreatic ducts express the α, βI--, δ-, ε-, η-, θ-, ζ- and μ-isoforms of PKC. In microperfused ducts, luminal H2DIDS (0.5 mM) caused intracellular pH to alkalinize and, like SP, inhibited basal and secretin- stimulated HCO3- secretion. SP did not inhibit secretion further when H2DIDS was present in the lumen, suggesting that SP and H2DIDS both inhibit the activity of an anion exchanger on the luminal membrane of the duct cell. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study in- vestigated the interaction between L-type Ca[sup 2+] current (ICaL) and Ca[sup 2+] release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Na[sup +, sub o]:K[sup +, sub I]) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca[sup 2+] current, TTX-sensitive ICa (ICaTTX), or "slip mode" Ca[sup 2+] conductance via INa+ were not responsible for low-voltage SRCR. However, L-type Ca[sup 2+] currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca[sup 2+] release from the SR at negative potentials is due to activation of L-type Ca[sup 2+] channels. [ABSTRACT FROM AUTHOR]

Inactivation of the L-type Ca[sup 2+] current (I[sub CaL]) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, I[sub CaL] of 82% of cells infused with Cs[sup +]based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of I[sub CaL] in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased I[sub CaL] by only ∼60% in these cells. Cells infused with either N-methyl-D-glucamine or K[sup +]-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased I[sub CaL] by ∼200% in these cells. In conclusion, we show that multiphasic inactivation of I[sub CaL] is due to Ca[sup 2+]-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs[sup +] seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K[sup +] in the pipette solution. [ABSTRACT FROM AUTHOR]

Examines the role of sodium/calcium exchange on intracellular sodium regulation in guinea pig detrusor smooth muscle. Reduction of extracellular sodium concentration; Evidence for sodium/calcium exchange; Generation of net calcium influx.