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PubMed2003

Endothelin-1 limits vascular smooth muscle beta-adrenergic receptor sensitivity by a PKC-dependent pathway.

Endothelin-1 reduces the chronotropic and inotropic effects of the beta-adrenoceptor agonist isoproterenol in rabbit isolated atria. Vascular interactions between endothelin-1 and isoproterenol have not been reported. Rings of the rabbit aorta without endothelium were mounted on myographs to measure isometric tension. Vessels were precontracted to similar levels with phenylephrine (30 micromol/L) or endothelin-1 (30 nmol/L). Relaxation to isoproterenol and forskolin were obtained. Vascular sensitivity (pD2) to isoproterenol was not different in the presence of endothelin-1 (7.6 +/- 0.3; n = 13) and phenylephrine (7.5 +/- 0.3; n = 11). The maximal relaxation (Emax) however, was doubled (P < 0.05) by endothelin-1 (42 +/- 5%), as compared with phenylephrine (23 +/- 4%). In the presence of endothelin-1, chelerythrine (protein kinase C inhibitor; 10 micromol/L) increased (P < 0.05) vascular sensitivity to isoproterenol (8.6 +/- 0.4, n = 7), but had no influence on the Emax. In contrast, in the presence of phenylephrine, pD2 was unaffected by chelerythrine, whereas the Emax to isoproterenol was increased (P < 0.05; 50 +/- 4%, n = 8). Vascular sensitivity and Emax to forskolin were similar in the presence of endothelin-1 and phenylephrine. In conclusion, endothelin-1 reduces vascular sensitivity to isoproterenol in a PKC-dependent pathway. The permissive effect of endothelin-1 appears to directly target the beta-adrenoceptor/G protein complex upstream of adenylate cyclase.

Alkaloids↗
PubMed2003

Converting enzyme inhibitor temocaprilat prevents high glucose-mediated suppression of human aortic endothelial cell proliferation.

We examined the involvement of the oxidative stress in high glucose-induced suppression of human aortic endothelial cell proliferation. Chronic glucose treatment for 72 h concentration-dependently (5.6-22.2 mol/l) inhibited human coronary endothelial cell proliferation. Temocaprilat, an angiotensin-converting enzyme inhibitor, at 10 nmol/l to 1 micromol/l inhibited high glucose (22.2 mmol/l)-mediated suppression of human aortic endothelial cell proliferation. Temocaprilat at 1 micromol/l inhibited high glucose-induced membrane-bound protein kinase C activity in human aortic endothelial cells. The protein kinase C inhibitors calphostin C 100 nmol/l or chelerythrine 1 micromol/l inhibited high glucose-mediated suppression of human aortic endothelial cell proliferation. Chronic high glucose treatment for 72 h increased intracellular oxidative stress, directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by temocaprilat 10 nmol/l to 1 micromol/l. Bradykinin B2 receptor antagonist icatibant 100 nmol/l significantly reduced the action of temocaprilat; whereas bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin 100 nmol/l had no effect. These findings suggest that high glucose inhibits human aortic endothelial cell proliferation and that the angiotensin-converting enzyme inhibitor temocaprilat inhibits high glucose-mediated suppression of human aortic endothelial cell proliferation, possibly through suppression of protein kinase C, bradykinin B2 receptors and oxidative stress.

Alkaloids↗
PubMed1998

Cytotoxic effects of sphingolipids as single or multi-modality agents on human melanoma and soft tissue sarcoma in vitro.

We evaluated the cytotoxic effects of a cell-permeable ceramide (Cer), N-hexanoyl-D-sphingosine (C6-Cer) and of two related sphingoid bases, sphingosine (So) and dihydrosphingosine (sphinganine; Sa) on human melanoma cell lines and on soft tissue sarcoma lines recently established from fresh surgical biopsy specimens. These cell lines ranged from high susceptibility (939 melanoma) to strong resistance (A2058 melanoma and all three sarcomas) to tumour necrosis factor (TNF), an inducer of elevated intracellular Cer levels. However, all the cell lines demonstrated a dose-dependent susceptibility to C6-Cer with protracted cytotoxic kinetics, with the C8161 melanoma being the most sensitive and A2058 the least. Protein kinase C (PKC) antagonizes Cer-dependent apoptosis, and chelerythrine chloride, So and Sa, which inhibit PKC, caused extremely rapid cytotoxicity of melanoma cell lines, irrespective of their relative sensitivity to C6-Cer. So-mediated cytotoxicity was extensive even after only 90 min of treatment, within the time frame of limb perfusion. So and Sa only slightly potentiated the cytotoxic responses to TNF, C6-Cer or melphalan. Sphingolipid-driven intracellular pathways may offer opportunities for therapy of these tumours.

Alkaloids↗
PubMed2000

Chelerythrine chloride induces rapid polymorphonuclear leukocyte apoptosis through activation of caspase-3.

Polymorphonuclear leukocytes (PMN) play a primary role in the initiation and propagation of inflammatory responses. PMN apoptosis is a major mechanism associated with the resolution of inflammatory reactions. Understanding mechanisms associated with PMN apoptosis will be of critical value in the development of novel pharmacological treatment strategies for local and/or systemic inflammatory disorders. The present study demonstrates that chelerythrine chloride induces human PMN to undergo rapid and synchronous progression into the apoptotic process via a PKC-independent mechanism. The appearance of the morphological features of apoptosis in chelerythrine-treated PMN is preceded by a significant upregulation in caspase-3 activity. GM-CSF (a cytokine that protects PMN in several models of PMN apoptosis) does not protect PMN from chelerythrine chloride-induced apoptosis.

Alkaloids↗
PubMed2005

Effects of chronic exposure to ethanol on glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement.

BACKGROUND: Glutamate transporters (excitatory amino acid transporters, EAAT) regulate extracellular concentrations of glutamate, a major excitatory neurotransmitter. We reported that acute ethanol exposure increases the activity of a major neuronal EAAT, EAAT3. This effect is consistent with the general inhibitory effect of acute alcohol toxicity in the central nervous system (CNS). However, chronic ethanol exposure has CNS presentations different from acute alcohol toxicity. We hypothesize that chronic ethanol exposure will affect the EAAT3 activity differently from acute ethanol exposure. METHODS: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Oocytes were incubated with diluted ethanol for 24-96 hr. Using two-electrode voltage clamp, membrane currents were recorded after the application of L-glutamate. Responses were quantified by integration of the current trace and reported as microCoulombs (microC). RESULTS: Ethanol (10-100 mM) reduced EAAT3 activity in a time-dependent and reversible manner. After a 96 hr-incubation, the activity was significantly decreased compared to the control values at any concentrations tested in this study. Kinetic study demonstrated that a 96 hr-exposure to 50 mM ethanol significantly decreased Vmax (3.6 +/- 0.3 for control versus 2.6 +/- 0.3 microC for ethanol, n = 20, p < 0.05) but had no effect on Km (57.6 +/- 12.8 for control versus 51.8 +/- 16.3 microM for ethanol, n = 20, p > 0.05) of EAAT3 for glutamate. When ethanol (50 mM for 96 hr)-treated oocytes were incubated with phorbol-12-myrisate-13-acetate (50 or 100 nM for 10 min), ethanol-induced decrease in EAAT3 activity was abolished. Preincubation of the oocytes with 100 microM chelerythrine significantly decreased EAAT3 activity (1.00 +/- 0.08 for control versus 0.51 +/- 0.09 microC for chelerythrine group, n = 18-20, p < 0.05). However, there was no statistical difference among the chelerythrine, ethanol, or chelerythrine plus ethanol groups. Likewise, staurosporine (2 microM for 1 hr) significantly decreased EAAT3 activity and there was no statistical difference among the staurosporine, ethanol, or staurosporine plus ethanol groups. CONCLUSIONS: Our results show that chronic ethanol exposure decreases EAAT3 activity at clinically relevant concentrations and that this effect may be protein kinase C-dependent. Such an effect could be a neuroadaptive mechanism to overcome the inhibitory effect of ethanol on the excitatory neurotransmission.

Alkaloids↗
PubMed2005

Staurosporine inhibits voltage-dependent K+ current through a PKC-independent mechanism in isolated coronary arterial smooth muscle cells.

We examined the effects of the protein kinase C (PKC) inhibitor staurosporine (ST) on voltage-dependent K (KV) channels in rabbit coronary arterial smooth muscle cells. ST inhibited the KV current in a dose-dependent manner with a Kd value of 1.3 microM. The inhibition of the KV current by ST was voltage-dependent between -30 and +10 mV. The additive inhibition of the KV current by ST was voltage-dependent throughout the activation voltage range. The rate constants of association and dissociation of ST were 0.63 microM s and 0.92 s, respectively. ST produced use-dependent inhibition of the KV current. ST shifted the activation curve to more positive potentials but did not have any significant effect on the voltage dependence of the inactivation curve. ST did not have any significant effects on other types of K channel. Another PKC inhibitor, chelerythrine, and PKA inhibitor peptide (PKA-IP) had little effect on the KV current. These results suggest that ST interacts with KV channels that are in the closed state and that ST inhibits KV channels in the open state in a manner that is phosphorylation-independent and voltage-, time-, and use-dependent.

Algorithms↗
PubMed2004

Endothelin-1 enhances the activity of Na+/Ca2+ exchanger type 1 in renal epithelial cells.

The Na+/Ca2+ exchanger, an ion transporter across the plasma membrane, is considered to play a role in calcium reabsorption in the renal nephron. We found that endothelin-1 enhanced Na+/Ca2+ exchange activity in renal epithelial LLC-PK1 cells. Treatment with endothelin-1 increased concomitantly the phosphorylation of Na+/Ca2+ exchanger type 1 (NCX1). Chelerythrine and prolonged exposure to phorbol 12-myristate 13-acetate abolished the activation of NCX1 induced by endothelin-1. To further analyze the activation mechanism of NCX1 by endothelin-1, we examined the effects of endothelin-1 in LLC-PK1 cells expressing NCX1 with mutated exchanger inhibitory peptide regions, which have either no Na+-dependent inactivation (XIP-4YW) or accelerated inactivation (F223E). The exchange activities in LLC-PK1 cells expressing wild-type NCX1 or F223E were stimulated by endothelin-1, but not in cells expressing XIP- 4YW. These results suggest that endothelin-1 activates NCX1 in renal epithelial cells through the pathway of protein kinase C and the process related to Na-dependent inactivation.

Animals↗
PubMed2004

Differential interplay between protein kinase C and Rho-kinase in the endothelin-1-induced and pressure-induced contractions of rat posterior cerebral artery.

To clarify the involvement of protein kinase C and Rho-kinase in the contractile activation of cerebral artery in response to endothelin-1 and pressurization, rat posterior cerebral artery (outer diameter, 100-200 microm) was mounted in arteriograph, and the changes in cytosolic Ca2+ and vessel diameter were measured by video-microscopy in connection with an Argus 50 system. Endothelin-1 (10 nM) induced a tonic contraction with a slight increase in cytosolic Ca2+, which was mostly dependent on protein kinase C (chelerythrine sensitive). Intraluminal pressurization (60 mmHg) also produced contraction with a low cytosolic Ca2+, which was myogenic in nature and dependent on both protein kinase C and Rho-kinase (Y-27632 sensitive). The results suggest differential interplay between protein kinase C and Rho-kinase in the endothelin-1-induced and pressure-induced tonic phase of contractions in the rat posterior cerebral artery.

Amides↗
PubMed2006

Rho-kinase contributes to hypoxia/reoxygenation-induced cerebral endothelial dysfunction.

Hypoxia/reoxygenation (H/R) in vitro induced cerebral endothelial dysfunction is mediated by superoxide production. However, the intracellular pathways involved remain unclear. The present study was designed to investigate the involvement of Rho-kinase and its interaction with nitric oxide (NO) in cerebral endothelial dysfunction after H/R. Arterial diameter and intraluminal pressure were simultaneously measured in vitro on rat posterior cerebral arteries. Vascular NO production was determined by measuring stable NO metabolites nitrate/nitrite. H/R selectively inhibited cerebral vasodilation to the endothelium-dependent agonist acetylcholine (ACh, 0.01 to 10 micromol/L) and caused NO deficiency. H/R-impaired vasodilation to ACh was reversed by Y27632 (1 micromol/L), a specific inhibitor of Rho-kinase, but not by chelerythrine (1 micromol/L), a selective inhibitor of protein kinase C. Y27632 had no protective effect in the presence of N-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L), a specific endothelial NO synthase inhibitor. L-NAME (100 micromol/L) alone failed to modulate H/R-impaired vasodilation, so did L-arginine (3 mmol/L), a substrate for NO synthase. However, a stable NO donor diethylenetetra amine-NONOate (5 micromol/L) normalized H/R-impaired dilation to ACh. In conclusion, H/R-induced endothelial dysfunction is associated with activation of Rho-kinase-dependent pathway and NO deficiency. Pretreatment with either Y27632 or the stable NO donor profoundly prevented H/R-mediated cerebral endothelial dysfunction.

Acetylcholine↗
PubMed2006

Sanguinarine downregulates AT1a gene expression in a hypertensive rat model.

We studied the in vivo effects of sanguinarine in a hypertensive rat model and its effects on AT1a mRNA expression in kidney tissues. Rats received daily for 14 d sanguinarine 0.1 mg/kg (SangL) and 0.3 mg/kg (SangH), losartan 1 mg/kg by weight (Los), or DMSO (Con). Blood pressures were monitored regularly and urine volume and sodium concentration was measured on days 0, 7, and 14. On day 15, animals were anesthetized (sodium thiopentane, 50 mg/kg), blood samples for aldosterone levels were taken, and kidneys were removed for AT1a mRNA expression. Los and SangH groups showed reduced AT1a mRNA expressions by 4.22- and 5.9-fold, respectively. In the SangL group it was reduced by 2.7-fold. Decreases in systolic blood pressures mirrored decreases in AT1a mRNA expressions in all groups. Los and SangH groups showed reductions in systolic blood pressure of 12.3% and 19.3%, respectively, whereas in the SangL group, it was reduced by 8.07%. Urine output in the Los group increased (228% mean increase from days 0-14), whereas sodium excretion decreased by 69.6% (mean decrease from days 0-14). In the SangL and SangH groups, urine volumes increased significantly by 108.3% and 115% (mean increase from days 0-14), respectively. Urinary sodium excretion increased significantly by 60.9% in the SangH group. We concluded that sanguinarine reduces blood pressure in the Dahl rat because of decreased AT1 receptor expression and reduced aldosterone levels. The action of losartan on increased urinary volume and decreased sodium excretion may be attributed to reduced vasopressin secretion.

Aldosterone↗
PubMed2005

Modulation of tumor necrosis factor-alpha and oxidative stress through protein kinase C and P42/44 mitogen-activated protein kinase in lead increases lipopolysaccharide-induced liver damage in rats.

Lead (Pb) increases lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), lipid peroxidation (LPO), and liver damage. In this study, we investigated the role of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (MAPK) and the causal relationships between TNF-alpha, NO, and LPO in Pb-increased LPS-induced liver damage in rats. Treatment with PKC and p42/44 MAPK inhibitors significantly reduced Pb + LPS-induced NO, TNF-alpha, LPO, and liver damage, which was revealed by elevated serum levels of aspartate aminotransferase and alanine aminotransferase. Pb + LPS coexposure significantly increased phosphorylation of p42/44 MAPK and TNF-alpha expression in peripheral blood cells; however, exposure to Pb + LPS did not induce TNF-alpha, NO, or LPO production and p42/44 MAPK activation in the liver. Pentoxifylline, a TNF-alpha inhibitor, also reduced liver damage but did not alter NO or LPO in Pb + LPS-treated rats. Thus, Pb increased LPS-induced liver damage through PKC and p42/44 MAPK modulation of TNF-alpha and oxidative stress, but modulation of TNF-alpha did not affect NO or LPO in rats.

Alanine Transaminase↗
PubMed2005

Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment.

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.

Alcohol Oxidoreductases↗
PubMed1996

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.

Acetates↗
PubMed2001

Protopine hydrochloride.

Protopine hydrochloride (5,6,14,14a-tetrahydro-14a-hydroxy-7-methyl-8H-bis[1,3]benzodioxolo[5,6-a:4,5-g]quinolizinium chloride, C20H20NO5(+)-Cl(-)) is the salt of the isoquinoline alkaloid protopine. It is formed by the action of dilute hydrochloric acid on the protopine free base. The N-methyl and hydroxyl groups are in a trans configuration in the quinolizine ring and the central quinolizine N-C bond is unusually long [1.579 (2) A]. The crystal is a racemate.

Alkaloids↗
PubMed1991

The effect of sanguinarine on human peripheral blood neutrophil viability and functions.

Human polymorphonuclear cell (PMN) viability, morphology, adherence, chemotaxis, oxidative metabolism, degranulation and phagocytosis were evaluated following treatment with sanguinarine (SANG). SANG was noncytotoxic to PMNs at all concentrations tested (0.31-200 microM). SANG entered the PMNs rapidly without altering the membrane fluidity and localized in the nuclear matrix. SANG (1.56-6.21 microM) inhibited chemotaxis, chemokinesis and adhesion in a dose-dependent manner, with a complete inhibition at 6.2 microM concentration. Concentrations of SANG up to 1.56 microM did not affect PMN oxidative burst; however, higher concentrations were found to inhibit basal as well as PMA-induced superoxide anion generation. The effect of SANG was time- and dose-dependent, and could be reversed if the PMNs were exposed to 12.5 microM or lower concentrations of SANG for less than 5 min. Autologous serum increased the tolerance of PMNs to SANG. Exogenous Ca2+ or Mg2+ did not alter the SANG-mediated inhibition of PMN functions. Treatment of PMNs with 3.12 microM or higher concentrations of SANG also resulted in inhibition of PMN degranulation and phagocytosis. The results suggest that SANG-mediated inhibition of PMN functions, without cytolysis or resultant release of inflammatory mediators, may have clinical implications.

Alkaloids↗
PubMed1997

Substance P modulates sensory action potentials in the lamprey via a protein kinase C-mediated reduction of a 4-aminopyridine-sensitive potassium conductance.

We have examined the effects of the tachykinin substance P on the action potential of lamprey mechanosensory dorsal cells. Substance P increased the spike duration and reduced the afterhyperpolarization. These effects were mimicked by stimulation of the dorsal root, which contains tachykinin-like immunoreactive fibres. The tachykinin antagonist spantide II blocked the effects of both substance P and dorsal root stimulation. The spike broadening was voltage-dependent, and was due to the reduction of a 4-aminopyridine-sensitive potassium conductance. The spike broadening was mimicked by G-protein activators and blocked by the G-protein inhibitor GDPbetaS. Pertussis toxin did not block the effects of substance P. The spike broadening was blocked by the protein kinase C and cAMP-dependent protein kinase inhibitor H7, and by the specific protein kinase C antagonist chelerythrine, but not by the cAMP and cGMP-dependent protein kinase inhibitor H8. The phorbol ester phorbol 12,13-dibutyrate mimicked and blocked the effects of substance P, supporting the role of protein kinase C in the spike modulation. The adenylate cyclase activator forskolin and the cAMP agonist SpcAMPs mimicked but did not block the effects of substance P on the spike duration, suggesting that protein kinase A also modulates the dorsal cell action potential, but that substance P acts independently of this pathway. Substance P also increased the excitability of the dorsal cells. This effect was blocked by 4-AP, PDBu and chelerythrine, but not by H8, suggesting that the increase in excitability shares the same intracellular and effector pathways as the spike broadening.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2005

Secretion of ATP from Schwann cells in response to uridine triphosphate.

The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y(2) and P2Y(4) receptor agonist, stimulated ATP release from Schwann cells in a dose-dependent manner with an ED(50) of 0.24 microm. UTP-stimulated ATP release occurs through P2Y(2) receptors as it was blocked by suramin which inhibits P2Y(2) but not P2Y(4) receptors. Furthermore, positive immunostaining of P2Y(2) receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y(4) receptors, did not significantly stimulate ATP release. UTP-stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP(3) formation inhibitor lithium chloride, the cell membrane-permeable Ca(2+) chelator BAPTA-AM and the endoplasmic reticulum Ca(2+)-dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi-complex inhibitor brefeldin A, microtubule disruption with nocodazole, F-actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP-stimulated ATP release is mediated by activation of P2Y(2) receptors that initiate an IP(3)-Ca(2+) cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.

Adenosine Triphosphate↗
PubMed1997

Stimulation of alpha 1-adrenoceptors and protein kinase C-mediated activation of ecto-5'-nucleotidase in rat hearts in vivo.

1. To determine whether protein kinase C (PKC)-mediated activation of ecto-5'-nucleotidase would increase interstitial adenosine concentrations in the rat heart in vivo, we made use of the microdialysis technique and a flexibly mounted probe, which was implanted in the left ventricular myocardium and perfused with Tyrode solution. 2. The baseline level of dialysate adenosine was 0.51 +/- 0.09 microM (n = 16). Perfusion of adenosine 5'-monophosphate (AMP, 100 microM) through the probe increased the dialysate adenosine concentration markedly to 9.25 +/- 0.46 microM (n = 15). alpha, beta-Methyleneadenosine 5'-diphosphate (AOPCP, 100 microM), an inhibitor of ecto-5'-nucleotidase, abolished the AMP-induced increase in dialysate adenosine, but did not affect the baseline level of adenosine. These observations suggest that the dialysate adenosine obtained during the perfusion with AMP, but not the baseline levels of adenosine, originated from the dephosphorylation of AMP by ecto-5'-nucleotidase. Thus, the level of adenosine measured during AMP perfusion gives an index of the activity of ecto-5'-nucleotidase in the tissue. 3. Noradrenaline (10 microM) increased the adenosine concentration measured in the presence of 100 microM AMP (i.e. the activity of ecto-5'-nucleotidase) by 38.7 +/- 9.6% (n = 5, P < 0.05), an increase which was inhibited by an antagonist of the alpha 1-adrenoceptor (prazosin, 50 microM) or of PKC (chelerythrine, 10 microM). Further application of either the alpha 1-adrenoceptor agonist methoxamine (100 microM) or the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol (DOG, 100 microM) also increased the adenosine concentration by 35.1 +/- 10.0% (n = 6, P < 0.05) or 40.6 +/- 8.3% (n = 5, P < 0.05), respectively. 4. The presence of okadaic acid (50 microM), an inhibitor of protein phosphatase, enhanced the noradrenaline-induced increase in adenosine concentration by 112.4 +/- 35.9% (n = 4, P < 0.05), to a level significantly (P < 0.05) greater than the increase caused by noradrenaline alone (38.7 +/- 9.6%). 5. These data provide the first evidence that alpha 1-adrenoceptor stimulation and the subsequent activation of PKC can increase adenosine concentrations in interstitial spaces of ventricular muscle in vivo, through activation of endogenous ecto-5'-nucleotidase.

5'-Nucleotidase↗