Induction of pacemaker currents by DA-9701, a prokinetic agent, in interstitial cells of Cajal from murine small intestine

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca²⁺-free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-β-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca²⁺ influx, phospholipase C activation, and Ca²⁺ release from internal storage in a G protein-independent and protein kinase C-independent manner.     

Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na⁺- free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a phospholipase C inhibitor, or thapsigargin, a Ca²⁺-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca²⁺ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca²⁺-release through neurotensin receptor1.     

A biophysically based mathematical model of unitary potential activity in interstitial cells of Cajal

Unitary potential (UP) depolarizations are the basic intracellular events responsible for pacemaker activity in interstitial cells of Cajal (ICCs), and are generated at intracellular sites termed “pacemaker units”. In this study, we present a mathematical model of the transmembrane ion flows and intracellular Ca²⁺ dynamics from a single ICC pacemaker unit acting at near-resting membrane potential. This model quantitatively formalizes the framework of a novel ICC pacemaking mechanism that has recently been proposed. Model simulations produce spontaneously rhythmic UP depolarizations with an amplitude of ∼3 mV at a frequency of 0.05 Hz. The model predicts that the main inward currents, carried by a Ca²⁺-inhibited nonselective cation conductance, are activated by depletion of sub-plasma-membrane [Ca²⁺] caused by sarcoendoplasmic reticulum calcium ATPase Ca²⁺ sequestration. Furthermore, pacemaker activity predicted by our model persists under simulated voltage clamp and is independent of [IP₃] oscillations. The model presented here provides a basis to quantitatively analyze UP depolarizations and the biophysical mechanisms underlying their production.     

Effects of sphingosine-1-phosphate on pacemaker activity of interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P_1,3,4,5 agonist) and SEW 2871 (an S1P₁ agonist) had no effects on pacemaker activity. Suramin (an S1P₃ antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P₂ antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P₂ in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH₂-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca²⁺-free solution or thapsigargin (a Ca²⁺-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca²⁺ ([Ca²⁺]_i) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca²⁺]_i oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P₂ via regulation of external and internal Ca²⁺ and mitogenactivated protein kinase activation.     

Biophysically Based Mathematical Modeling of Interstitial Cells of Cajal Slow Wave Activity Generated from a Discrete Unitary Potential Basis

Spontaneously rhythmic pacemaker activity produced by interstitial cells of Cajal (ICC) is the result of the entrainment of unitary potential depolarizations generated at intracellular sites termed pacemaker units. In this study, we present a mathematical modeling framework that quantitatively represents the transmembrane ion flows and intracellular Ca²⁺ dynamics from a single ICC operating over the physiological membrane potential range. The mathematical model presented here extends our recently developed biophysically based pacemaker unit modeling framework by including mechanisms necessary for coordinating unitary potential events, such as a T-Type Ca²⁺ current, V_m-dependent K⁺ currents, and global Ca²⁺ diffusion. Model simulations produce spontaneously rhythmic slow wave depolarizations with an amplitude of 65 mV at a frequency of 17.4 cpm. Our model predicts that activity at the spatial scale of the pacemaker unit is fundamental for ICC slow wave generation, and Ca²⁺ influx from activation of the T-Type Ca²⁺ current is required for unitary potential entrainment. These results suggest that intracellular Ca²⁺ levels, particularly in the region local to the mitochondria and endoplasmic reticulum, significantly influence pacing frequency and synchronization of pacemaker unit discharge. Moreover, numerical investigations show that our ICC model is capable of qualitatively replicating a wide range of experimental observations.     

The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca²⁺ from intracellular stores and activation of Ca²⁺-activated Cl⁻ channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca²⁺ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na⁺/Ca²⁺ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca²⁺ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca²⁺ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca²⁺ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na⁺/Ca²⁺ exchange inhibitor, CGP-37157 inhibited Ca²⁺ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca²⁺ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca²⁺ (Ca_V3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced Ca_V3.2 currents. CCCP blocked Ca_V3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca²⁺ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.     

Pacemaking activity is regulated by membrane stretch via the CICR pathway in cultured interstitial cells of Cajal from murine intestine

Membrane stretch is an important stimulus in gastrointestinal (GI) motility regulation, but the relationship between membrane stretch and the pacemaking activity of GI smooth muscle is poorly understood. We examined the effect of intestinal distension on slow waves and the effect of membrane stretch on pacemaker currents in cultured intestinal interstitial cells of Cajal (ICCs) from murine small intestine. At organ level, intestinal distension significantly increased amplitude of slow and fast waves, and enhanced frequencies of fast but not slow waves. At the cellular level, membrane stretch-induced by hyposmotic cell swelling (MSHC) depolarized membrane potential and activated large inward holding current, but suppressed amplitude of pacemaker potential or pacemaking current. External Ca²⁺-free solution abolished pacemaker current and blocked MSHC-induced inward holding current. However, a sustained inward holding current was activated and the amplitude of pacemaker current was increased by high ethylene glycol tetraacetic acid (EGTA) in pipette. Then MSHC also potentiated the inward holding current. MSHC significantly increased amplitude of rhythmic Ca²⁺ transients and basal intracellular Ca²⁺ concentration ([Ca²⁺]_i). 2-APB blocked both pacemaker current and Ca²⁺ transients but did not alter the effect of MSHC on pacemaker current and Ca²⁺ transients. In contrast, ryanodine inhibited Ca²⁺ transients but not pacemaker current, and completely blocked MSHC-induced inward holding current and MSHC-induced increase of basal [Ca²⁺]_i. These results suggest that intestinal distension potentiates intestinal motility by increasing the amplitude of slow waves. Membrane stretch potentiates pacemaking activity via releasing Ca²⁺ from calcium-induced calcium release (CICR) in cultured intestinal ICCs.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro

Specialized olfactory receptor neurons in insects respond to species-specific sex pheromones with transient rises in inositol trisphosphate and by opening pheromone-dependent cation channels. These channels resemble cation channels which are directly or indirectly Ca²⁺-dependent. But there appear to be no internal Ca²⁺ stores in the outer dendrite where the olfactory transduction cascade is thought to start. Hence, it remains to be determined whether an influx of external Ca²⁺ precedes pheromone-dependent cation currents. Patch clamp measurements in cultured olfactory receptor neurons from Manduca sexta reveal that a transient inward current precedes pheromone-dependent cation currents. A transient inositol trisphosphate-dependent Ca²⁺ current, also preceding cation currents with the characteristics of pheromone-dependent cation currents, shares properties with the transient pheromone-dependent current. These results match the biochemical measurements with the electrophysiological data obtained in insect olfactory receptor neurons.Abbreviations ORNs Olfactory receptor neurons - IP₃ Inositol-1,4,5-trisphosphate - I_t Transient pheromone-dependent current - I_ir Transient IP₃-dependent current     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K⁺ stimulated ⁴⁵Ca²⁺ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca²⁺ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca²⁺ uptake occurred within 15 s of stimulation by carbachol or elevated K⁺ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K⁺ was similar to that of Ca²⁺ uptake. There was a close correlation between Ca²⁺ uptake and catecholamine secretion at various concentrations of Ca²⁺. The concentration dependencies for inhibition of both processes by Mg²⁺ or Cd²⁺ were similar. Ca²⁺ uptake saturated with increasing Ca²⁺ concentrations, with an apparent K_m for both carbachol-induced and elevated K⁺-induced Ca²⁺ uptake of approximately 2 mM. The Ca²⁺ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca²⁺ entry and a presumed increase in cytosolic Ca²⁺ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca²⁺- dependent processes associated with catecholamine secretion. Ca²⁺ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca²⁺ channels.     

Interaction between the M2- and M3-Receptor Subtypes in Muscarinic Electrical and Mechanical Responses of Intestinal Smooth Muscles

In various gastrointestinal smooth muscles, two different muscarinic receptor subtypes, M₂ and M₃, are expressed; these receptors are the target for the parasympathetic neurotransmitter acetylcholine. Although the number of M₂ receptors is much greater than that of M₃ receptors, the functional role of the former receptor subtype has yet to be fully defined, since pharmacological analyses of the contractile responses to acetylcholine and other muscarinic agonists have revealed that such responses are mediated extensively by the minor M₃ subtype. The M₃ receptor links to Ca²⁺ store release, and the released Ca²⁺ ions may contribute to the contraction. However, many studies indicated the importance of Ca²⁺ influx through voltage-gated Ca²⁺ channels, rather than Ca²⁺ release, in muscarinic contractions, since the contractile responses are markedly inhibited by Ca²⁺ channel blockers. The major M₂ receptors link to the opening of cationic channels leading to the membrane depolarization, which in turn activates voltage-gated Ca²⁺ channels. Thus, there should be somewhere a point of contact between the M₃- and M₂-mediated signal transductions, as if M₃ receptor stimulation is connected with membrane depolarization. Our electrophysiological and pharmacological findings suggest that the M₂-mediated cationic channel opening and a resulting increase in the membrane electrical activity are the primary mechanism for mediating the contractile response to muscarinic agonists. An allosteric interaction between M₂ and M₃ receptors such that M₃ activation intensifies the M₂/cation channel pathway may account at least in part for the failure of many previous analyses to detect M₂ participation in the contractile responses to full agonists.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

Differential sensitivity to nickel and SK&F96365 of second messengerm operated and receptormoperated calcium channels in rat submandibular ductal cells

The intracellular concentration of calcium ([Ca²⁺]_i) of rat submandibular ductal cells was measured with the intracellular fluorescent dye Fura-2. Carbachol (100 μM) and ATP (1 mM) both increased the [Ca²⁺]_j. The late response to ATP was blocked by 0.5 mM Ni²⁺. This concentration of Ni²⁺ also blocked the increase of the [Ca²⁺]_i and the uptake of manganese and calcium in response to 2′- and 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP, 100 μM), a specific agonist of P2X receptors from salivary glands. The increase of the [Ca²⁺]_i in response to 2-methylthioadenosine 5′-triphosphate (2-McSATP, 100 μM) a specific P2Y agonist in salivary glands or to a muscarinic agonist (carbachol) was not affected by 0.5 mM Ni²⁺. Only higher concentrations of Ni²⁺ (in the millimolar range) inhibited the uptake of extracellular calcium in response to carbachol. SK&F 96365, a blocker of store-operated calcium channels, inhibited the uptake of extracellular calcium in response to carbachol without affecting the response to BzATP. It is concluded that at low concentrations (below 0.5 mM), Ni²⁺ inhibits the non-specific cation channel coupled to P2X receptors. The uptake of extracellular calcium by store-operated calcium channels is inhibited by higher concentrations of Ni²⁺ and by SK&F96365.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a_1S subunit of the skeletal L-type channel (Ca_v1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Ca_v1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Ca_v1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Ca_v1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Ca_V channels on physiological processes that extend beyond channel gating and permeability.     

In vivo adaptative regulation of muscarinic receptors and muscarinic stimulation-induced Ca2+ mobilization during short-term heat exposure in rat parotid glands

1. Adaptation of muscarinic receptors (MR)—muscarinic stimulation—induced intracellular Ca²⁺ mobilization during short-heat exposure (33°C).2. Heat-exposure for 48 hr decreased the carbachol (CCh)-stimulated cytosolic C²⁺ concentration increase.3. The number of MR on cell surface increased transiently at 24 hr with a subsequent decrease at 48 hr.4. CCh-stimulated inositol trisphosphate (IP₃) formation decreased at 48 hr.5. In saponin-permeabilized cells, 1,4,5-IP₃-induced ⁴⁵Ca²⁺ release decreased at 24 hr.6. The data suggest that the adaptation for increased muscarinic stimulation occurs at IP₃ generating sites as well as at intracellular IP₃ receptor sites during heat exposure.     

Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

Dissecting out the Complex Ca2+-Mediated Phenylephrine-Induced Contractions of Mouse Aortic Segments

L-type Ca²⁺ channel (VGCC) mediated Ca²⁺ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (V_m) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca²⁺ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca²⁺ release and the tonic contraction determined by Ca²⁺ influx. The latter component involves both Ca²⁺ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca²⁺ influx by small variations of the VSMC V_m causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca²⁺ release from non-contractile sarcoplasmic reticulum Ca²⁺ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca²⁺ release form contractile or non-contractile Ca²⁺ stores with concomitant Ca²⁺ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.