Addition Of CCh Research Articles

Ivabradine inhibits carbachol-induced contractions of isolated rat urinary bladder.

Overactive bladder (OAB), a symptom syndrome defined as urgency, is a common clinical condition, which sometimes cannot be satisfactorily treated with current medications in every subject; therefore, alternatives are needed. The aim of this in vitro study was to investigate the effects of ivabradine, a selective pacemaker If current inhibitor, on agonist-induced isometric contractions of the bladder smooth muscles. Urinary bladder strips were isolated from adult male Wistar rats and suspended in a tissue bath containing physiological solution. The strips were contracted by bath applications of carbachol (CCh, 1 μM). Ivabradine (30 μM, 60 μM or 90 μM) was added to the tissue bath either prior to or after the application of the agonist, and the resulting contractile activity was compared to the preceding contractile activity. The amplitude and area under force-time curves (AUFC) of the isometric contractions were evaluated. The addition of CCh caused a marked stimulation of contractile force in isolated urinary bladder strips, which was significantly inhibited by ivabradine, both in terms of peak amplitude (29% ±3%, 20% ±6% and 18% ±6% by 30 μM, 60 μM and 90 μM ivabradine, respectively) and AUFC (47% ±5.5%, 35% ±8% and 35% ±6% by 30 μM, 60 μM and 90 μM ivabradine, respectively; n = 7 for each). Pre-treatment with ivabradine (10 μM) significantly attenuated the contractile response to CCh (1 μM; mean peak amplitude from 1493 ±216 mg to 680 ±95 mg; p < 0.003; n = 7). The results of this in vitro study demonstrated that ivabradine inhibits cholinergic agonistinduced bladder contractions, which means that in the future ivabradine may be used in OAB treatment.

Competitive supramolecular interaction of carbachol and berberine with cucurbit[7]uril and its analytical application

Carbachol (CCH) is non-fluorescent in aqueous solution. This property makes its determination through direct fluorescent method impossible. Berberine (BER) exhibits very weak fluorescence emissions in aqueous solution. However, In acidic medium and room temperature, BER can react with CB[7] to form stable complex and the fluorescence intensity of the complex was greatly enhanced. The dramatic quenching of the fluorescence intensity of CB[7]–BER complex was observed with the addition of CCH. The competitive supramolecular interaction between CCH and BER for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by spectrofluorometry, 1H NMR, and molecular modeling calculation. Based on the significant quenching of the supramolecular complex fluorescence intensity, a simple, rapid, and sensitive fluorescence probe method for the determination of CCH in pharmaceutical dosage forms and in biological fluids was developed. The linear range of the method was from 0.01 μg mL− 1 to 1.5 μg mL− 1. The limit of detection was 0.003 μg mL− 1. This shows that the proposed method has promising potential for quality control, forensic identification, drug abuse or therapeutic monitoring, pharmacokinetics, and clinical application.

Protein Kinase C-dependent Phosphorylation of Transient Receptor Potential Canonical 6 (TRPC6) on Serine 448 Causes Channel Inhibition

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).

Vascular endothelial cells and smooth muscle cells mediate carbachol‐induced hepatocyte proliferation via muscarinic receptors and IP3/PKC signaling cascades

An acetylcholine (ACh) agonist, carbachol (Cch), causes hepatocytes to proliferate in the presence of hepatic nonparenchymal cells (HNPCs). To identify the HNPCs and ACh receptor subtypes involved in carbachol-induced hepatocyte proliferation (CIHP), we examined two types of vascular cells as candidates for HNPCs mediating CIHP in cocultures of hepatocytes using the Transwell filter insert. In the coculture with vascular smooth muscle cells (VSMCs) or endothelial cells (VECs), but not in the monoculture, 72 h treatment with Cch significantly increased the numbers of hepatocytes. The results suggest that both VSMCs and VECs are involved in CIHP through soluble factors secreted from these cells. Interestingly, coculture with VECs, but not with VSMCs, markedly increased the number of hepatocytes, even in the absence of Cch. Cell proliferation assays using an analogue of thymidine, bromodeoxyuridine (BrdU), demonstrated that the hepatocytes in both cocultures transiently replicated their chromosomes 12 h after Cch administration. Blocking the muscarinic type 1 ACh receptor (M1), M3/5, intracellular inositol triphosphate (IP3) receptor, or protein kinase C (PKC) pathways inhibited VSMC-mediated CIHP, whereas blocking the M3/5, IP3 receptor, or PKC pathways inhibited VEC-mediated CIHP. Co-culturing hepatocytes with both types of vascular cells markedly increased their albumin content, but addition of Cch had no effect. In conclusion, VSMCs among vascular cells mediate CIHP through M1, M3/5, and IP3/PKC signal transduction pathways, whereas VECs do so through M3/5, and IP3/PKC pathways.

Peculiarities of phospholipase C-dependent release of CA2+ from intracellular stores upon activation of choline and purine receptors in myocytes of the guinea-pig small intestine

Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca2+]i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 µM carbachol, CCh, as well as 10.0 and 100.0 µM ATP) induced a rise in the [Ca2+]i. Carbachol evoked an increase in the [Ca2+]i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca2+]i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca2+]i in myocytes. The absence of the slow component in the [Ca2+]i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca2+ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca2+]i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 µM), completely blocked rises in the [Ca2+]i induced by both CCh and ATP; therefore, the release of Ca2+ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca2+ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles.

Segmental heterogeneity of electrogenic secretions in human ascending colon and rectum

We have attempted to ascertain putative segmental differences in the secretory responses of the human ascending colon and rectum. From the mucosal biopsy samples of two segments, the short-circuit current (I(sc)) and tissue resistance (R(te)) were compared under control conditions, as well as after the induction of secretion, using a modified Ussing chamber. We also performed semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to detect and quantify transport proteins. The spontaneous I(sc) in the ascending colon was found to be greater than that in the rectum (P<0.01), whereas isobutylmethylxanthine/forskolin and carbachol (CCh) induced a greater rise in I(sc) in the rectum than in the ascending colon (P<0.05). When coupled with indomethacin pretreatment, the increase in Delta I(sc) after the addition of CCh and forskolin was significant as compared to that observed without pretreatment (P<0.05). However, in the rectum, the secretory response to CCh and forskolin was abolished to a significant degree by indomethacin (P<0.05). Moreover, these indomethacin-induced changes were reversed by the addition of PGE2. Upon semiquantitative RT-PCR analysis, the amounts of cystic fibrosis transmembrane regulator, KCNQ1, and CLCA1 mRNAs were not found to be different between the two segments. There was a clear segmental heterogeneity with regard to electrogenic secretion in the human colon, and this difference can be explained by differences in the ascending colon and rectum.

Imaging of intracellular Ca2+ waves induced by muscarinic receptor stimulation in rat parotid acinar cells

Changes in cytosolic Ca2+ concentration ([Ca2+]i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca2+, the increase in [Ca2+]i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca2+ wave toward the basolateral region. The [Ca2+]i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca2+ was eliminated from the extracellular medium, the Ca2+ wave was a result of Ca2+ release from intracellular stores. The magnitude and velocity of the Ca2+ wave decreased with decreasing concentration of CCh, and the increase in [Ca2+]i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca2+ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca2+ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca2+ pump, caused a slow and homogeneous increase in [Ca2+]i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca2+]i suggesting that the inositol-1,4,5-trisphosphate (InsP3) and ThG-sensitive Ca2+ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP3 is essential to trigger the Ca2+ wave and that the development of the Ca2+ wave may be attributed to regional differences in InsP3 sensitivity of Ca2+ stores. The agonist-induced Ca2+ wave is probably a general phenomenon in exocrine acinar cells.

Effect of carbachol on phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis, and its modulation by isoproterenol in rabbit corneal epithelial cells.

The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation by isoproterenol were investigated in SV40-adenovirus transformed rabbit corneal epithelial cells (RCEC). When examined under light microscope, these cells exhibited a cobblestone-like appearance typical of the corneal epithelial cells grown in primary culture. Addition of CCh (0.1 mM) for 30 min to RCEC, prelabeled with 32Pi, decreased the radioactivity in phosphatidylinositol 4-phosphate and PIP2 by 15 and 27%, respectively, and concomitantly increased the radioactivity in phosphatidylinositol and phosphatidic acid by 14 and 38%, respectively. When the concentration of CCh was increased to 1 mM, the changes in radioactivity were even more pronounced. Addition of CCh (0.1 mM) to the cells, prelabeled with myo[3H]inositol, increased the accumulation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3) by 115%, indicating stimulation of PLC-mediated PIP2 hydrolysis. Similar increases were also observed in [3H]InsP1 and [3H]InsP2. The effects of CCh on inositol phosphate accumulation were time- and dose-dependent, and were inhibited by atropine (10 microM), suggesting that the observed effects of CCh were mediated by activation of muscarinic cholinergic receptors. The effects of CCh were antagonized more potently by 4-diphenylacetoxy N-methyl-piperidine than by pirenzepine, indicating that the muscarinic receptors involved in PLC activation are probably of M3 type. By Western immunoblotting analysis with various anti-PLC antibodies, the RCEC were shown to contain PLC gamma 1 and PLC delta 1 in the soluble fraction and PLC beta 1 in the microsomal fraction. Addition of isoproterenol to RCEC, increased cAMP both in a time- and dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of Capacitative Ca2+ Entry by Serine/Threonine Phosphatase Inhibitors in Rat Parotid Acinar Cells

The effects of three serine/threonine protein phosphatase inhibitors, calyculin-A, tautomycin and okadaic acid, on the Ca2+ entry across the plasma membrane was studied in Fura-2-loaded rat parotid acinar cells. These protein phosphatase inhibitors did not affect the peak elevation of cytosolic free Ca2+ concentration ([Ca2+]i) just after stimulation with the muscarinic agonist carbachol (CCh), but they suppressed the sustained increase in [Ca2+]i. In the absence of extracellular Ca2+, CCh produced a transient increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, and this increase in [Ca2+]i was unaffected by the phosphatase inhibitors. When Ca2+ was added to the external medium after the transient [Ca2+]i response, the increase in [Ca2+]i in the cells treated with the phosphatase inhibitors was significantly smaller than that in the control cells, indicating that the Ca2+ entry was reduced. Similar suppression of Ca2+ entry by the phosphatase inhibitors was observed when intracellular Ca2+ stores were previously depleted by the microsomal Ca2+-ATPase inhibitor thapsigargin (TG). In addition, the phosphatase inhibitors reduced the Mn2+ (Ca2+ surrogate) influx following the addition of CCh or TG. The enhancement of Ca2+ entry by the protein kinase inhibitor staurosporine was significantly attenuated by the phosphatase inhibitors. These results suggest that the phosphatase inhibitors suppressed the Ca2+ entry mechanism activated by depletion of intracellular Ca2+ stores in rat parotid acinar cells. The capacitative Ca2+ entry may be regulated by protein phosphorylation/dephosphorylation.

Staurosporine enhances Ca 2+ entry induced by depletion of intracellular Ca 2+ stores in rat parotid acinar cells

The effect of staurosporine on the Ca 2+ signalling induced by the muscarinic receptor agonist carbachol (CCh) was studied in Fura 2-loaded rat parotid acinar cells. At concentrations > 1 nM, staurosporine dose-dependently enhanced the sustained increase in cytosolic free Ca 2+ concentration ([Ca 2+] i), but did not affect the peak [Ca 2+] i seen just after stimulation. The enhancement of the sustained increase in [Ca 2+] i was not attenuated by the protein kinase C activator, 4β-phorbol 12-myristate 13-acetate, and not mimicked by another inhibitor of Protein kinase C, K-252a, suggesting that the effect of staurosporine on the CCh-induced Ca 2+ signalling may be due to a mechanism independent of the inhibitory action on protein kinase C. Staurosporine also enhanced the increases in [Ca 2+] i induced by the microsomal Ca 2+-ATPase inhibitor thapsigargin (TG) and the Ca 2+ ionophore ionomycin (Iono). When the cells were stimulated by CCh, TG, or Iono in the absence of extracellular Ca 2+, a transient increase in [Ca 2+] i due to Ca 2+ release from intracellular stores was observed. This increase in [Ca 2+] i was unaffected by preincubation with staurosporine. However, when Ca 2+ was added to the extracellular medium after [Ca 2+] i had returned to the resting level, the increase in [Ca 2+] i was significantly enhanced by staurosporine.In addition, staurosporine accelerated the Mn 2+ influx following the addition of CCh, TG, or Iono. These results suggest that staurosporine modulates the Ca 2+ entry system activated by depletion of intracellular Ca 2+ stores in rat parotid acinar cells.

Identification of Phosphoinositide-specific Phospholipase C-β1 and GTP-binding Protein, G qα, in Bovine Iris Sphincter Membranes: Characteristics of the Phospholipase and its Coupling to Cholinergic Muscarinic Receptors

Previously, we have established that treatment of iris sphincter smooth muscle with carbachol (CCh) results in increased phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3) and in muscle contraction. To throw more light on the mechanism of muscarinic stimulation of PLC in this tissue we have investigated the properties of this enzyme and its regulation by GTP analogs and protein phosphorylation in bovine iris sphincter membranes. The data obtained can be summarized as follows: (1) the presence of PLC-β 1 and a GTP-binding protein. G qα, was detected in the microsomal (membrane) fraction by anti-PLC β1 and anti-G qα antibodies, respectively. The membrane PLC hydrolysed exogenously added PIP 2 and this hydrolysis was increased dose-dependently by Ca 2+ (1-10 μM) but the enzyme activity was inhibited by Mg 2+. (2) Addition of guanosine 5'-O-thiotriphosphate (GTPγS, 0·1 μM) to the membrane fraction increased PIP 2 hydrolysis by 30%, whereas addition of CCh (10 μM) was without effect. However, when added together, CCh and GTPγS increased PIP 2 hydrolysis by 46%. This effect was significantly inhibited by atropine and by the anti-PLC-β1 and anti-G qα antibodies. (3)Removal of PLC-β1 from the membranes with 2 m KCl resulted in a significant reduction of the CCh-induced PIP 2 hydrolysis, and this effect of the muscarinic agonist was restored when the membrane fraction was supplemented with PLC-β1 purified from bovine brain. (4) CCh-stimulated PIP 2 hydrolysis was significantly reduced in membrane fractions prepared from iris sphincter pretreated with the β-adrenergic agonist, isoproterenol. Thus, it can be concluded that in bovine iris sphincter: (a) muscarinic stimulation of PLC-β1, and subsequently PIP 2 hydrolysis, is mediated through the G-protein. G qα; and (b) that phosphorylation of G qα and/or muscarinic receptor by protein kinase A could be the mechanism through which isoproterenol inhibits CCh-stimulated hydrolysis of PIP 2 and muscle contraction in this tissue.

Neurotransmitter-induced changes in arachidonate incorporation into rat brain phospholipids: pharmacological manipulations of receptors, presynaptic uptake, mao activity and deacylation-reacylation processes

The incorporation of [(3)H]arachidonic acid ([(3)H]AA) into phospholipids (PL) of rat brain, was studied in cerebral cortex slices in the presence and absence of norepinephrine (NE), serotonin (5-HT) and carbamylcholine (CCH). Both NE and 5-HT produced a concentration-dependent effect of stimulating [(3)H]AA incorporation into phosphatidylinositol (PI) while attenuating incorporation into other PL. Addition of CCH had no apparent effect. The beta-adrenergic agonist, isoproterenol, had an effect similar to that seen with equimolar concentrations of NE, whereas the alpha(1) agonist, phenylephrine, or the alpha(2) agonist, clonidine, did not produce significant changes. However, application of the NE-receptor blockers, propranolol or prazosin, in the presence of NE, did not modify the NE-induced effects. Similarly, the 5-HT-receptor blockers, methysergide or ketanserine, failed to modify the 5-HT-induced effects, indicating that the neurotransmitter-produced changes may not be receptor mediated. Manipulations of the NE or 5-HT reuptake systems by imipramine (IMI) or desipramine (DMI) had a small additive effect on the neurotransmitter-produced changes in [(3)H]AA incorporation, suggesting that a functional presynaptic reuptake system is not required for the NE or 5-HT-produced effects. The possibility that the NE or 5-HT effects involve the oxidative metabolism of the monoamines by MAO was also investigated. The MAO inhibitors tranylcypromine and pargyline had no appreciable effect on the neurotransmitter-induced changes in [(3)H]AA incorporation whereas clorgyline clearly reduced the increase in [(3)H]AA incorporation into PI seen in the presence of NE or 5-HT, but this clorgyline effect may not be related to its activity as MAO inhibitor. The phospholipase A(2) inhibitor mepacrine had no significant effect on the NE-produced increase in [(3)H]AA incorporation into PI, but it antagonized the NE-produced decrease in [(3)H]AA incorporation into PC. Delta-9-Tetrahydrocannabinol, which acts as acyltransferase inhibitor, antagonized the NE-produced increase in [(3)H]AA incorporation into PI without appreciably influencing the NE-produced decrease in [(3)H]AA incorporation into PC. These findings suggest that the neurotransmitter-produced increase in [(3)H]AA incorporation into PI is mediated by stimulation of a specific lyso-PI arachidonyl transferase. The neurotransmitter effects on arachidonate incorporation may have physiological significance in view of the importance of processes of deacylation and reacylation of membrane PL in regulating the function of neuronal membranes.