Frequency Of Pacemaker Potentials Research Articles

Effect of adenosine-1 receptor activation on pacemaker activity of interstitial cells of Cajal from mouse colon.

Adenosine plays an important role on gastrointestinal (GI) motility through adenosine receptors. Interstitial cells of Cajal (ICC) are pacemaker cells that regulate GI smooth muscle activity. The functional role and its signal mechanism of adenosine on the pacemaker activity were investigated using whole-cell patch clamp, RT-PCR, and intracellular Ca2+-imaging with ICC from mouse colon. Adenosine depolarized the membrane potentials and increased the pacemaker potential frequency, which was blocked by a selective A1-receptor antagonist, but not A2a-, A2b, or A3-receptor antagonist. A selective A1 receptor agonist represented similar effects as those of adenosine and mRNA transcript of A1-receptor was expressed in ICC. The adenosine-induced effects were blocked by phospholipase C (PLC) and a Ca2+-ATPase inhibitor. Adenosine increased spontaneous intracellular Ca2+ oscillations, as seen fluo4/AM. Both hyperpolarization-activated cyclic nucleotide (HCN) channel inhibitors and adenylate cyclase inhibitors blocked the adenosine-induced effects. And adenosine increased the basal cellular adenylate cyclase activity in colonic ICC. However, adenosine and adenylate cyclase inhibitors did not show any influence on pacemaker activity in small intestinal ICC for a comparison with that of the small intestine. These results suggest adenosine modulates the pacemaker potentials by acting HCN channels- and intracellular Ca2+- dependent mechanisms through A1-receptor. Therefore, adenosine may be a therapeutic target in colonic motility disorders.

Regulation of Intracellular Calcium by Endoplasmic Reticulum Proteins in Small Intestinal Interstitial Cells of Cajal.

Background/AimsWe investigated the role of representative endoplasmic reticulum proteins, stromal interaction molecule 1 (STIM1), and store-operated calcium entry-associated regulatory factor (SARAF) in pacemaker activity in cultured interstitial cells of Cajal (ICCs) isolated from mouse small intestine.MethodsThe whole-cell patch clamp technique applied for intracellular calcium ions ([Ca2+]i) analysis with STIM1 or SARAF overexpressed cultured ICCs from mouse small intestine.ResultsIn the current-clamping mode, cultured ICCs displayed spontaneous pacemaker potentials. External carbachol exposure produced tonic membrane depolarization in the current-clamp mode, which recovered within a few seconds into normal pacemaker potentials. In STIM1-overexpressing cultured ICCs pacemaker potential frequency was increased, and in SARAF-overexpressing ICCs pacemaker potential frequency was strongly inhibited. The application of gadolinium (a non-selective cation channel inhibitor) or a Ca2+-free solution to understand Orai channel involvement abolished the generation of pacemaker potentials. When recording intracellular Ca2+ concentration with Fluo 3-AM, STIM1-overexpressing ICCs showed an increased number of spontaneous intracellular Ca2+ oscillations. However, SARAF-overexpressing ICCs showed fewer spontaneous intracellular Ca2+ oscillations.ConclusionEndoplasmic reticulum proteins modulated the frequency of pacemaker activity in ICCs, and levels of STIM1 and SARAF may determine slow wave patterns in the gastrointestinal tract.

Effects of Ca2+-Activated Cl- Channel ANO1inhibitors on Pacemaker Activity in Interstitial Cells of Cajal

Background/Aims: Anoctamin1 (Ca²⁺-activated Cl^- channel, ANO1) is a specific marker of the interstitial cells of Cajal (ICC) in the gastrointestinal tract, and are candidate proteins that can function as pacemaker channels. Recently, novel selective ANO1 inhibitors were discovered and used to study Ca²⁺-activated Cl^- channels. Therefore, to investigate whether ANO1 channels function as pacemaker channels, selective ANO1 inhibitors were tested with respect to the pacemaker potentials in ICC. Methods: Whole-cell patch-clamp recording, RT-PCR, and intracellular Ca²⁺ ([Ca²⁺]_i) imaging were performed in cultured ICC obtained from mice. Results: Though CaCCinh-A01 (5 µM), T16Ainh-A01 (5 µM), and MONNA (5 µM) (selective ANO1 inhibitors) blocked the generation of pacemaker potentials in colonic ICC, they did not do so in small intestinal ICC. Though nifulmic acid (10 µM) and DIDS (10 µM) (classical Ca²⁺-activated Cl^- channel inhibitors) also had no effect in small intestinal ICC, they suppressed the generation of pacemaker potentials in colonic ICC. In addition, knockdown of ANO1 reduced the pacemaker potential frequency in colonic ICC alone. Though ANO1 inhibitors suppressed [Ca²⁺]_i oscillations in colonic ICC, they did not do so in small intestinal ICC. T-type Ca²⁺ channels were expressed in the both the small intestinal and colonic ICC, but mibefradil (5 µM) and NiCl₂ (30 µM) (T-type Ca²⁺ channel inhibitors) inhibited the generation of pacemaker potentials in colonic ICC alone. Conclusion: These results indicate that though ANO1 and T-type Ca²⁺ channels participate in generating pacemaker potentials in colonic ICC, they do not do so in small intestinal ICC. Therefore, the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon.

ATP-sensitive K+ channels maintain resting membrane potential in interstitial cells of Cajal from the mouse colon

To investigate the role of ATP-sensitive K+(KATP) channels on pacemaker activity in interstitial cells of Cajal (ICC), whole-cell patch clamping, RT-PCR, and intracellular Ca2+([Ca2+]i) imaging were performed in cultured colonic ICC. Pinacidil (a K+ channel opener) hyperpolarized the membrane and inhibited the generation of pacemaker potential, and this effect was reversed by glibenclamide (a KATP channel blocker). RT-PCR showed that Kir 6.1 and SUR2B were expressed in Ano-1 positive colonic ICC. Glibenclamide depolarized the membrane and increased pacemaker potential frequency. However, 5-hydroxydecanoic acid (a mitochondrial KATP channel blocker) had no effects on pacemaker potentials. Phorbol 12-myristate 13-acetate (PMA; a protein kinase C activator) blocked the pinacidil-induced effects, and PMA alone depolarized the membrane and increased pacemaker potential frequency. Cell-permeable 8-bromo-cyclic AMP also increased pacemaker potential frequency. Recordings of spontaneous intracellular Ca2+([Ca2+]i) oscillations showed that glibenclamide increased the frequency of [Ca2+]i oscillations. In small intestinal ICC, glibenclamide alone did not alter the generation of pacemaker potentials, and Kir 6.2 and SUR2B were expressed in Ano-1 positive ICC. Therefore, KATP channels in colonic ICC are activated in resting state and play an important role in maintaining resting membrane potential.

Regulation of the Pacemaker Activity of Colonic Interstitial Cells of Cajal by Protease-Activated Receptors: Involvement of Hyperpolarization-Activated Cyclic Nucleotide Channels

Background and Purpose: The exact mechanism of protease-activated receptors (PARs) on pacemaker activity of interstitial cells of Cajal (ICCs) has not been reported. We investigated the effects on pacemaker activity by the activation of PARs and its signal mechanisms in colonic ICCs. Methods: The whole-cell patch-clamp technique, RT-PCR and Ca²⁺ imaging were used in cultured ICCs from mouse colon. Results: PAR-1 and PAR-2 were expressed in Ano-1 positive ICCs. TFLLR-NH₂ (a PAR-1 agonist) and trypsin (a PAR-2 agonist) depolarized the membrane and increased the pacemaker potential frequency. U-73122 (a phospholipase C (PLC) inhibitor) and thapsigargin (a Ca²⁺ ATPase inhibitor) suppressed the TFLLR-NH₂- and trypsin-induced effects on pacemaker potential. TFLLR-NH₂ and trypsin also increased intracellular Ca²⁺ ([Ca²⁺]_i) intensity with increasing of Ca²⁺ oscillations. Genistein (a tyrosine kinase inhibitor), SP600125 (a JNK inhibitor), CsCl, ZD7288, clonidine (hyperpolarization-activated cyclic nucleotide (HCN) channel blockers), SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) suppressed the increased pacemaker potential frequency without effects on depolarization of the membrane induced by TFLLR-NH₂ and trypsin. Conclusion: These results suggest that activation of PAR-1 and PAR-2 modulates the pacemaker activity of colonic ICCs through the PLC-dependent [Ca²⁺]_i release pathway. The increased pacemaker potential frequency by PAR-1 and PAR-2 was also dependent on tyrosine kinase, JNK, and HCN activation.

Regulatory Roles of Endogenous Mitogen-Activated Protein Kinases and Tyrosine Kinases in the Pacemaker Activity of Colonic Interstitial Cells of Cajal

Background and Purpose: Mitogen-activated protein (MAP) and tyrosine kinases play an important role in regulating smooth muscle contraction of the gastrointestinal (GI) tract. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. Thus, the role of MAP and tyrosine kinases on the pacemaker potentials of colonic ICCs was investigated. Methods: Cultured ICCs were prepared from mice colons, and their pacemaker potentials were recorded using whole-cell patch clamping. Results: In current-clamping mode, colonic ICCs displayed spontaneous pacemaker potentials. SB203580 (a p38 MAP kinase inhibitor), SP600125 (a c-jun NH₂-terminal kinase (JNK) inhibitor), genistein and herbimycin A (tyrosine kinase inhibitors) blocked the generation of pacemaker potentials. However, PD98059 (a p42/44 MAP kinase inhibitor) had no effects on pacemaker potentials. LY-294002 (phosphoinositide 3-kinase inhibitor) also reduced the pacemaker potential frequency but calphostin C and chelerythrine (protein kinase C inhibitors) had no effects. However, PD98059, SB203589, SP600125, genistein, herbimycin A, LY-294002, and calphostin C had no effect on normal pacemaker activity in small intestinal ICCs. Conclusions: Endogenous p38 MAP kinases, JNKs, tyrosine kinases, and PI3-kinases participate in the generation of pacemaker potentials in colonic ICCs but not in ICCs of the small intestine.

The traditional herbal medicine, Ge-Gen-Tang, inhibits pacemaker potentials by nitric oxide/cGMP dependent ATP-sensitive K+ channels in cultured interstitial cells of Cajal from mouse small intestine

Ethnopharmacological relevanceGe-Gen-Tang (GGT) is a traditional Chinese medicinal formula composed of Puerariae radix (Pueraria lobata Ohwi), Ephedrae Herba (Ephedra sinica Stapf), Cinnamomi Ramulus (Cinnamomum cassia Blume), Paeoniae Radix (Paeonia lactiflora Pallas), Glycyrrhizae Radix preparata (Glycyrrhiza uralensis Fischer), Zingiberis Rhizoma (Zingiber officinale Roscoe), and Zizyphi Fructus (Ziziphus jujuba Mill. var. inermis Rehder) and is widely used to ameoliorate the symptoms of gastrointestinal (GI) disorders related to diarrhea and intestinal mucosal immunity and for anti-cold, antipyretic and analgesic in Eastern Asia. Aim of the studyInterstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. We investigated the effects of GGT on pacemaker potentials in cultured ICCs from the mouse small intestine, and sought to identify the receptors and the action mechanisms involved. Materials and methodsEnzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed on within 12h after culture. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. Intracellular Ca2+ ([Ca2+]i) increase was studied in cultured ICCs using fura-2AM. All of the experiments were performed at 30–32°C. ResultsUnder the current clamping mode, GGT decreased the amplitude and frequency of pacemaker potentials; however, these effects were blocked by intracellular GDPβS, a G-protein inhibitor, and glibenclamide, a specific ATP-sensitive K+ channels blocker. Prazosin (α1-adrenoceptor antagonist) and butoxamine (β2-adrenoceptor antagonist) did not block the GGT-induced effects, whereas atenolol (β1-adrenoceptor antagonist) blocked the GGT-induced effects. Also, yohimbine (α2-adrenoceptor antagonist) partially blocked the GGT-induced effects. Pretreatment with SQ-22536, an adenylate cyclase inhibitor, did not block the GGT-induced effects, whereas pretreatment with ODQ, a guanylate cyclase inhibitor, or L-NAME, an inhibitor of nitric oxide (NO) synthase, did. Additionally, [Ca2+]i analysis showed that GGT decreased [Ca2+]i. ConclusionThese results suggest that GGT inhibits pacemaker potentials in ICCs in a G protein-, cGMP- and NO-dependent manner through stimulation of α2 and β1-adrenoceptors.

Functional effects of β3-adrenoceptor on pacemaker activity in interstitial cells of Cajal from the mouse colon

We investigated the presence of β3-adrenoceptor and its functional effects on pacemaker potentials in colonic interstitial cells of Cajal (ICCs) from mice. The whole-cell patch clamp technique was used to record pacemaker potentials in cultured ICCs and reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA transcript levels β-adrenoceptors. The β3-adrenoceptor agonist, BRL37344, reduced the frequency of pacemaker potentials in a concentration-dependent manner. The inhibitory effects of BRL37344 were blocked by the pretreatment of propranolol, a nonspecific β-adrenoceptor antagonist, but not by the selective β1-adrenoceptor antagonist atenolol and the selective β2-adrenoceptor antagonist butoxamine. β3-adrenoceptor antagonists SR59230A and L748337 blocked the inhibitory effects of BRL37344. RT-PCR revealed mRNA transcripts of β1- and β3-adrenoceptor, but not β2-adrenoceptor, in c-kit- and Ano-1-positive colonic ICCs. The K+ channel blockers tetraethylammonium, apamin, and glibenclamide did not block the effects of BRL37344. Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME), an NO synthase inhibitor, and chelerythrine, a protein kinase C inhibitor, also did not block the effects of BRL37344. Noradrenaline mimicked the effects of BRL37344 in colonic ICCs. However, the inhibitory effects of noradrenaline on pacemaker potentials were blocked only by pretreatment with atenolol but not by butoxamine, SR59230A, or L748337. In small intestinal ICCs, BRL37344 had no effect on pacemaker potentials and mRNA transcripts of β1-and β2-adrenoceptor, but not β3-adrenoceptor were detected. These results suggest that β3-adrenoceptors are present in colonic ICCs and may play a role in regulating gastrointestinal motility by the inhibition of pacemaker potentials.

Pituitary Adenylate Cyclase-activating Polypeptide Inhibits Pacemaker Activity of Colonic Interstitial Cells of Cajal.

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K+ channel blocker). However, neither NG-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K+ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

The possible roles of hyperpolarization-activated cyclic nucleotide channels in regulating pacemaker activity in colonic interstitial cells of Cajal

BackgroundHyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs.MethodsWe performed whole-cell patch clamp, RT-PCR, and Ca2+-imaging in cultured ICCs from mouse mid colon.ResultsSQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca2+ [Ca2+]i oscillations, rolipram and 8-bromo-cAMP increased [Ca2+]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca2+]i oscillations.ConclusionsHCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP

Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC(50) value of ∼30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ∼0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.

Direct Inhibition of Cardiac Hyperpolarization-Activated Cyclic Nucleotide–Gated Pacemaker Channels by Clonidine

Inhibition of cardiac sympathetic tone represents an important strategy for treatment of cardiovascular disease, including arrhythmia, coronary heart disease, and chronic heart failure. Activation of presynaptic alpha2-adrenoceptors is the most widely accepted mechanism of action of the antisympathetic drug clonidine; however, other target proteins have been postulated to contribute to the in vivo actions of clonidine. To test whether clonidine elicits pharmacological effects independent of alpha2-adrenoceptors, we have generated mice with a targeted deletion of all 3 alpha2-adrenoceptor subtypes (alpha2ABC-/-). Alpha2ABC-/- mice were completely unresponsive to the analgesic and hypnotic effects of clonidine; however, clonidine significantly lowered heart rate in alpha2ABC-/- mice by up to 150 bpm. Clonidine-induced bradycardia in conscious alpha2ABC-/- mice was 32.3% (10 microg/kg) and 26.6% (100 microg/kg) of the effect in wild-type mice. A similar bradycardic effect of clonidine was observed in isolated spontaneously beating right atria from alpha2ABC-knockout and wild-type mice. Clonidine inhibited the native pacemaker current (I(f)) in isolated sinoatrial node pacemaker cells and the I(f)-generating hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 and HCN4 channels in transfected HEK293 cells. As a consequence of blocking I(f), clonidine reduced the slope of the diastolic depolarization and the frequency of pacemaker potentials in sinoatrial node cells from wild-type and alpha2ABC-knockout mice. Direct inhibition of cardiac HCN pacemaker channels contributes to the bradycardic effects of clonidine gene-targeted mice in vivo, and thus, clonidine-like drugs represent novel structures for future HCN channel inhibitors.

Effects of temperature on pacemaker potentials in the mouse small intestine

The effects of temperature on the generation of pacemaker potentials recorded from myenteric interstitial cells of Cajal (ICC-MY) distributed in the mouse small intestine were investigated using intracellular recording techniques. In response to increasing temperatures in the range of 26-40 degrees C, the frequency and maximum rate of rise (dV/dt (max)) of pacemaker potentials were increased while their duration was decreased. The resting membrane potential and amplitude of the pacemaker potentials were not affected by change in temperature. Elevation of temperature decreased the amplitude, duration, and rise time of unitary potentials generated spontaneously during intervals between the pacemaker potentials. Metabolic inhibition (KCN and iodoacetic acid) decreased the frequency of pacemaker potentials with no alteration to the amplitude and dV/dt (max). Cyclopiazonic acid (3 muM), an inhibitor of the internal Ca(2+) pump, abolished pacemaker potentials in low-temperature conditions (<29 degrees C) but not at high-temperature conditions (>38 degrees C). These results suggest that the primary and plateau components of pacemaker potentials have different temperature sensitivities: the primary component is highly temperature-sensitive and is activated at higher temperatures, while the plateau component is formed by activation of temperature-insensitive mechanisms. The results also suggest that the mitochondria-induced intracellular Ca(2+) handling system seems to be involved in the initiation of the generation of pacemaker potentials but not in their configuration.

Pacemaker frequency is increased by sodium nitroprusside in the guinea pig gastric antrum.

In the guinea pig gastric antrum, the effects of sodium nitroprusside (SNP), an NO donor, on pacemaker potentials were investigated in the presence of nifedipine. The pacemaker potentials consisted of primary and plateau components; SNP (> 1 microM) increased the frequency of occurrence of these pacemaker potentials, while inhibiting the plateau component. 1H-[1,2,4]-Oxadiazole [4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase, had no effect on the excitatory actions of SNP on the frequency of pacemaker potentials. Other types of NO donor, (+/-)-S-nitroso-N-acetylpenicillamine, 3-morpholino-sydnonimine and 8-bromoguanosine 3'5'-cyclic monophosphate had no excitatory effect on pacemaker activity. Forskolin, an activator of adenylate cyclase, or 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid, an inhibitor of the Ca(2+)-activated Cl(-) channel, strongly attenuated the generation of pacemaker potentials, and SNP added in the presence of these chemicals restored the generation of pacemaker potentials. The pacemaker potentials evoked by SNP were abolished in low-Ca(2+) solution or by membrane depolarization with high-K(+) solution. The SNP-induced generation of pacemaker potentials was not prevented by cyclopiazonic acid, an inhibitor of internal Ca(2+)-ATPase, but was limited to a transient burst by iodoacetic acid, an inhibitor of glycolysis, carbonyl cyanide m-chlorophenyl-hydrazone, a mitochondrial protonophore, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular Ca(2+) chelator. These results suggest that the SNP-induced increase in the frequency of pacemaker potentials is related to the elevated intracellular Ca(2+) concentrations due to release from mitochondria, and these actions may be independent of the activation of guanylate cyclase.

Properties of unitary potentials recorded from myenteric interstitial cells of Cajal distributed in the guinea-pig gastric antrum.

Intracellular recordings were made from myenteric interstitial cells of Cajal (ICC-MY) distributed in the guinea-pig gastric antrum to investigate the properties of unitary potentials. In most cells studied, pacemaker potentials with initial fast transient and following plateau components were generated periodically, and intervals between the potentials were quiescent. However, there were few cells (less than 5% of cells examined) which showed discharge of unitary potentials spontaneously in the intervals between pacemaker potentials. The amplitude and frequency of unitary potentials appeared to be random variables, as observed in isolated circular smooth muscle bundles of the guinea-pig gastric antrum. BAPTA-AM (an intracellular Ca2+ chelator) or papaverine (a non-selective phosphodiesterase inhibitor) reduced the discharge frequency of unitary potentials, with associated decrease in the frequency of pacemaker potentials. These agents finally abolished both unitary potentials and pacemaker potentials. In preparations showing no detectable generation of unitary potentials, depolarization of the membrane with high-K solution ([K+]o = 10.6 mM) elicited generation of unitary potentials during intervals between pacemaker potentials. Pinacidil (an opener of K(ATP)-channels) hyperpolarized the membrane and increased the frequency and amplitude of unitary potentials with no alteration to the relationship between the amplitudes of unitary potentials and their half-widths. These results suggest that the elevation of intracellular Ca2+ concentration is causally related to the generation of unitary potentials in pacemaker cells. They are consistent with the proposition that the depolarization produced by a burst of unitary potentials triggers the primary component of pacemaker potentials in ICC-MY, which induces a release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive internal stores and then activates Ca2+-sensitive Cl- -channels to form the plateau component. Similarities and differences in unitary potentials between circular muscle and pacemaker cells are discussed.

CGRP inhibition of electromechanical coupling in the guinea-pig isolated renal pelvis.

We aimed at studying the mechanism(s) of the inhibitory effect exerted by calcitonin gene-related peptide (CGRP) on the spontaneous activity of the guinea-pig isolated renal pelvis. In organ bath experiments, CGRP (1-100 nM) produced a concentration-dependent (EC50 8 nM) partial inhibition (Emax about 35% inhibition of motility index) of spontaneous contractions. The potassium (K) channel opener, cromakalim (3-10 microM) promptly suppressed the spontaneous contractions in a glibenclamide-(10 microM) sensitive manner. Glibenclamide (10 microM) did not affect the inhibitory action of CGRP. The calcium (Ca) channel agonist, Bay K 8644 (1 microM), markedly enhanced the spontaneous activity of the renal pelvis and reduced the inhibitory effect of CGRP. The protein kinase A inhibitors Rp-cAMPS (300 microM), H8 (100 microM) and H89 (10 microM), and the blockers of intracellular Ca handling by sarcoplasmic reticulum, ryanodine (100 microM) and thapsigargin (1 microM) did not affect the response to CGRP. The response to CGRP was likewise unaffected by the nitric oxide synthase inhibitor, L-nitroarginine (30 microM) and by the protein kinase G inhibitor, KT5823 (3 microM). Furthermore, the inhibitory action of CGRP was not modified by lowering the extracellular concentration of K (from 5.9 to 1.2 mM) nor by increasing (from 2.5 to 3.75 mM) or decreasing (from 2.5 to 0.25 mM) the extracellular Ca concentration. Replacement of 80% glucose with 2-deoxyglucose (2-DOG) reduced the amplitude of spontaneous contractions, both in the absence and presence of 10 microM glibenclamide. In the presence of 2-DOG, the inhibitory action of CGRP was enhanced at a similar extent, either in the absence or presence of glibenclamide. In sucrose gap, the effect of CGRP (0.1 microM for 5 min) was separately analyzed in the proximal (close to the kidney) and distal (close to the ureter) regions of the renal pelvis. Both preparations discharged spontaneous (pacemaker) action potentials having different shape, duration and frequently. CGRP had no effect on pacemaker potentials in the proximal renal pelvis while producing about 30% reduction of the frequency of pacemaker potentials and motility index in the distal renal pelvis. Cromakalim (3 microM) abolished pacemaker potentials in both regions of the renal pelvis. In conjunction with the results of previous studies in the guinea-pig ureter, the present findings document the existence of remarkable regional differences in the effector mechanisms initiated by CGRP receptor occupancy in the guinea-pig pyeloureteral tract. CGRP appears to be inherently unable to activate glibenclamide-sensitive K channels in the guinea-pig renal pelvis, a mechanism which is central for its ability to suppress latent pacemakers in the ureter. Within the renal pelvis, the sensitivity to the inhibitory effect of CGRP appears in the more distal region, from which an 'ureter-like' action potential is recorded.

Pharmacological characterization of cholinoreceptors of cardiac ganglion cells of crustaceans

1. 1. Acetylcholine applied to neurons of the heart ganglion of Squilla mantis causes slight depolarization, an increased slope and frequency of pacemaker potentials and an increased number of action potentials in each burst. The result is an increased rate and amplitude of heart beats. 2. 2. Isolated and opened hearts of lobsters ( Homarus americanus) continue to beat. ACh applied to the heart muscle is without effect. When applied to the heart ganglion directly, it causes an immediate increase in rate and amplitude of heart beats. 3. 3. The nature of the cholinoreceptors on heart ganglion cells was explored in the crayfish, Astacus leptodactylus, by perfusing the semi-isolated heart and recording its contractions. Supplementary experiments were performed with a similar technique on hearts of the crab, Eriphia spinifrons. 4. 4. ACh and muscarinic agonists are powerful excitatory agents; of these methacholine is at least as effective as ACh; methylfurmethide, muscarine and carbachol are nearly as effective. Nicotine, on the other hand causes excitation only at high concentration and is four orders of magnitude less effective than muscarine. A number of other agents were also tested. 5. 5. Muscarinic antagonists are effective blockers; nicotinic blockers, on the other hand, have a very low potency. This is especially true for bis-quarternary compounds. 6. 6. The significance of the presence of typical muscarinic cholinoreceptors in crustacean ganglion cells is discussed, especially in the light of Michelson's concept of the evolution of cholinoreceptor structure.