Release of dopamine and norepinephrine by hypoxia from PC-12 cells

We examined the effects of hypoxia on the release of dopamine(DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cellsand assessed the involvement ofCa²⁺ and protein kinases instimulus-secretion coupling. Catecholamine release was monitored bymicrovoltammetry using a carbon fiber electrode as well as by HPLCcoupled with electrochemical detection (ECD). Microvoltammetricanalysis showed that hypoxia-induced catecholamine secretion(PO₂ ofmedium ~40 mmHg) occurred within 1 min after the onset of thestimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysisrevealed that, at any level of PO₂, therelease of NE was greater than the release of DA. In contrast, inresponse to K⁺ (80 mM), DA releasewas ~11-fold greater than NE release. The magnitude ofhypoxia-induced NE and DA releases depended on the passage, source, andculture conditions of the PC-12 cells. Omission of extracellularCa²⁺ or addition of voltage-gatedCa²⁺ channel blockers attenuatedhypoxia-induced release of both DA and NE to a similarextent. Protein kinase inhibitors, staurosporine (200 nM) andbisindolylmaleimide I (2 µM), on the other hand, attenuatedhypoxia-induced NE release more than DA release. However, proteinkinase inhibitors had no significant effect onK⁺-induced NE and DA releases.These results demonstrate that hypoxia releases catecholamines fromPC-12 cells and that, for a given change inPO₂, NErelease is greater than DA release. It is suggested that proteinkinases are involved in the enhanced release of NE during hypoxia.

     

Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells

Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA-induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson-like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12-36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100-500 microM) and KCN (100-500 microM) induced a concentration- and time-dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end-labeling) staining, was also concentration- and time-dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA-induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 microM) and/or KCN (100 microM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60-70% of the cells from DA- and DA plus KCN-induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA- and KCN-induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA-induced apoptosis. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide-mediated oxidative stress as an initiator of apoptosis.     

Cyanide induces protein kinase C translocation: Blockade by NMDA antagonists

Activation and translocation of protein kinase C (PKC) during KCN-induced histotoxic hypoxia was studied in rat brain slices prepared from cerebellum, hippocampus, and cortex. Treatment with 1–10 mM KCN produced a significant increase in PKC translocation and enzyme activity in the particulate fraction of cerebellar and hippocampal slices. In cortical slices, PKC activity was not affected by cyanide treatment. The membrane-associated PKC activity reached a maximum 30 minutes after incubation with KCN and remained elevated up to 60 minutes in both the hippocampus and cerebellum. Pretreatment with MK-801 and APV, specific NMDA receptor antagonists, blocked the cyanide-stimulated translocation in the hippocampus and cerebellum, whereas CNQX, an AMPA/kainate receptor antagonist, did not alter the response. These results demonstrate that cyanide stimulates PKC activation and translocation from the cytosol to membranes in select brain areas and NMDA receptor activation mediates this process.     

Modulation of [3H]dopamine release from rabbit retina

The calcium-dependent release of [3H]dopamine ([3H]DA) elicited by field stimulation or potassium is modulated through activation of stereoselective inhibitory DA autoreceptors of the D-2 subtype that are pharmacologically different from the D-1 DA receptor subtype linked to the stimulation of adenylate cyclase (EC 4.6.1.1). The D-2 DA autoreceptors appear to be endogenously activated by DA because DA receptor antagonists such as S-sulpiride increased the stimulation-evoked release of [3H]DA. Nanomolar concentrations of norepinephrine (NE) and epinephrine (E) inhibited in a concentration-dependent manner the electrical stimulation-evoked release of [3H]DA. The inhibitory effect of these catecholamines was not modified by S-sulpiride, which, on the contrary, selectively antagonized the inhibition of [3H]DA release elicited by exogenous DA. Phentolamine or (+/-)-propranolol did not affect the release of [3H]DA from rabbit retina. The alpha antagonist phentolamine competitively antagonized the inhibitory effect of both NE and E, which suggests that these catecholamines activate alpha receptors in retina. The decrease by catecholamines of the calcium-dependent release of [3H]DA appears not to involve beta adrenoceptors because their inhibitory effect was not modified by propranolol. Under identical experimental conditions (i.e., nomifensine, 30 microM), serotonin did not modify the stimulated release of [3H]DA. In conclusion, in the rabbit retina, DA autoreceptors of the D-2 subtype appear to modulate endogenously released DA whereas inhibitory presynaptic alpha receptors might be of pharmacological importance as sites of action for retinal or blood-borne catecholamines.     

Support for a physiological role of endogenous catecholamines in the stimulation of bovine luteal progesterone production

To determine if catecholamines were present in bovine luteal tissue, corpora lutea (CL) were obtained during the mid-luteal phase (Days 10-12) and the concentration of dopamine (DA) and norepinephrine (NE) was determined by high-performance liquid chromatography. Both DA and NE were detected in luteal tissue at mean concentrations of 41.9 +/- 5.73 and 10.2 +/- 2.51 ng/g for DA and NE, respectively. These concentrations represented a luteal content of 306.6 +/- 66.88 ng/CL for DA and 70.5 +/- 16.88 ng/CL for NE. In vitro, DA at concentrations of 1.0 mM to 0.01 mM stimulated the production of progesterone (P4, p less than 0.05). The response to DA was inhibited by propranolol (a beta-adrenergic receptor antagonist, p less than 0.05) but not by phentolamine, phenoxybenzamine (alpha-adrenergic receptor antagonists), or haloperidol (a DA receptor antagonist, p greater than 0.05). Neither L-tyrosine nor L-dopa altered P4 production (p greater than 0.05). Inhibition of DA beta-hydroxylase, the enzyme that catalyzes the conversion of DA to NE by FLA-63 blocked the DA-induced increases in luteal P4 production (p less than 0.05). These results demonstrate the existence of DA and NE in bovine luteal tissue and indicate that exogenous DA can be converted to NE in luteal tissue. The results support a physiological role for catecholamines in the stimulation of bovine luteal function.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Effect of protein kinase C inhibition on hypoxic pulmonary vasoconstriction

The current study was done to test the hypothesis that protein kinase C (PKC) inhibitors prevent the increase in pulmonary vascular resistance and compliance that occurs in isolated, blood-perfused dog lungs during hypoxia. Pulmonary vascular resistances and compliances were measured with vascular occlusion techniques. Hypoxia significantly increased pulmonary arterial resistance, pulmonary venous resistance, and pulmonary capillary pressure and decreased total vascular compliance by decreasing both microvascular and large-vessel compliances. The nonspecific PKC inhibitor staurosporine (10(-7) M), the specific PKC blocker calphostin C (10(-7) M), and the specific PKC isozyme blocker G?-6976 (10(-7) M) inhibited the effect of hypoxia on pulmonary vascular resistance and compliance. In addition, the PKC activator thymeleatoxin (THX; 10(-7) M) increased pulmonary vascular resistance and compliance in a manner similar to that in hypoxia, and the L-type voltage-dependent Ca(2+) channel blocker nifedipine (10(-6) M) inhibited the response to both THX and hypoxia. These results suggest that PKC inhibition blocks the hypoxic pressor response and that the pharmacological activation of PKC by THX mimics the hypoxic pulmonary vasoconstrictor response. In addition, L-type voltage-dependent Ca(2+) channel blockade may prevent the onset of the hypoxia- and PKC-induced vasoconstrictor response in the canine pulmonary vasculature.     

Effects of l-DOPA on the Concentrations of Free and Sulfoconjugated Cathecholamines in Plasma, Cerebrospinal Fluid, Urine, and Central and Peripheral Nervous System Tissues of the Rat

The effects of subcutaneous injection of L-beta-3,4-dihydroxyphenylalamine (L-DOPA) on the concentrations of the catecholamines and catecholamine sulfates in the central and peripheral nervous systems of the rat were studied. The results showed that free 3,4-dihydroxyphenylethylamine (DA, dopamine) increased rapidly and markedly in the hypothalamus and striatum after L-DOPA but DA sulfate did not change. Increased concentrations of DA sulfate were detected in the CSF and in the plasma, where it reached a concentration of 130.8 +/- 12.8 ng/ml at 2 h, seven times the level of free DA (19.1 +/- 2.9 ng/ml). In the kidney the ratio of DA sulfate to free DA was reversed in favor of free DA. Urine samples of L-DOPA-treated rats showed a higher increase of free DA than DA sulfate, but free norepinephrine (NE) and NE sulfate remained unchanged. Concentrations of free DA and free NE in the adrenal glands of L-DOPA-treated rats showed no change. Adrenal DA sulfate and NE sulfate were not detectable in the control and L-DOPA-treated rats, suggesting that the adrenal glands lack the capacity to take up or store catecholamines and their sulfate counterparts from the plasma.     

Role of Protein Kinase C α in Nerve Growth Factor-Induced Arachidonic Acid Release from PC12 Cells

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

Dopamine Release via Protein Kinase C Activation in the Fish Retina

Calcium-dependent phospholipid-sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activated PKC in the nanomolar range. A major 38-kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (greater than 15 min). Rod-bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (alpha/beta-subtype). The PKC antibody recognized a 78-kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium- and dose-dependent [3H]DA release during a short period (2 min), with the minimal effective dose being approximately 1 nM. Other phorbols having no tumor-promoting activity, such as 4 beta-phorbol and 4 alpha-phorbol 12,13-didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1-oleoyl-2-acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG- or TPA-induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free, glucuronide, and sulfate catecholamines in the rat: effect of hypoxia

The formation and excretion of conjugated catecholamines (CA) was studied in conscious rats after sympathetic stimulation by hypoxia (5.5-6% O2, 4 h). Hypoxia induced a rapid and intense increase of free epinephrine (E, X 12) and norepinephrine (NE, X 6) but only a limited enhancement of free dopamine (DA, X 2). Sulfate conjugates of E and NE had kinetics similar to the free forms, while glucuronides were only moderately and lately altered. In contrast to free and sulfated DA, DA glucuronide, the major plasma conjugate, was decreased (-25%). This result suggests that DA glucuronide, unlike other CA conjugates, is not related to detoxication but might supply a CA precursor. Urinary conjugates badly reflected plasma conjugates. In normoxic controls, CA conjugates prevailed in the plasma, whereas the free amines prevailed in the urine. Hypoxia increased mainly the excretion of E and NE glucuronide but not of the free amines. Urinary DA, free or conjugated, was decreased (-25%), a result in keeping with plasma DA glucuronide only. The poor relations between plasma and urine catecholamines pinpoint the importance of the kidney in CA handling.     

Cytosolic-free calcium and neurotransmitter release with decreased availability of glucose or oxygen

Exposing brain slices to reduced oxygen tensions or impairing their ability to utilize oxygen with KCN decreases acetylcholine (ACh) but increases dopamine (DA) and glutamate in the medium at the end of a release incubation. To determine if these changes are due to alterations in the presynaptic terminals, release from isolated nerve endings (i.e. synaptosomes) was determined during histotoxic hypoxia (KCN). KCN reduced potassium-stimulated synaptosomal ACh release and increased dopamine and glutamate release. Since several lines of evidence suggest that altered calcium homeostasis underlies these changes in release, the effects of reducing medium calcium concentrations from 2.3 to 0.1-mM were determined. In low calcium medium, KCN still increased dopamine and glutamate release, but had no effect on ACh release. Hypoxia increased cytosolic-free calcium in both the normal and low calcium medium, although the elevation was less in the low calcium medium. Thus, the effects of histotoxic hypoxia on cytosolic free calcium concentration paralleled those on glutamate and dopamine release. Reducing the glucose concentration of the medium also increased cytosolic-free calcium. The data are consistent with the hypothesis that hypoxia and hypoglycemia increase cytosolic-free calcium, which stimulates the release of dopamine and glutamate, whose excessive release may lead to subsequent cellular damage postsynaptically.Abbreviations (cps) counts per second - (FAM) fura-2 acetoxymethylester - (ACh) acetylcholine - (Ca_i) cytosolic free calcium concentration - (DMSO) dimethylsulphoxide - (DA) dopamine - (TES) N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid - (R_min) the ratio of the fluorescence of fura at 510 nm after excitation at 340 nm to that after excitation at 380 nm in the absence of calcium - (R_max) or to that in the presence of saturating calcium - (SNK) Student-Newman-Keuls     

Free and conjugated catecholamines and metabolites in cat urine after hypoxia

Catecholamine and metabolite excretion was studied in the cat after 6 h of 7.5% O2 hypoxia. Norepinephrine (NE) release from sympathetic nervous endings was strongly activated, whereas epinephrine (E) excretion was only slightly increased. A noteworthy result was the increase of dopamine (DA) and its metabolites [3-methoxytyramine (MT); 3,4-dihydroxyphenylacetic acid (DOPAC)] in urine samples. This increased release does not seem to originate from the central nervous system, but rather from peripheral dopaminergic structures; available knowledge on peripheral DA suggests that the hypoxia-induced DA release might be partly related to chemosensory or renal function. Indeed, in addition to enhanced DA and NE excretion, we observed an increase in sodium excretion that correlated with both DA and NE. Analysis of free and conjugated urinary metabolites showed that only free NE and both free and conjugated normetanephrine were increased in urine after hypoxic stress. Among DA metabolites, conjugated DOPAC was the main DA metabolite in the basal state and after hypoxia. Both the free and the conjugated forms of DA, MT, and DOPAC were increased by hypoxia.     

Catecholamines in larvae and juveniles of the prosobranch gastropod, Crepidula fornicata

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K+. Competent larvae contained 1.58 +/- 0.26 (S.E.M.) x 10(-2) pmol DOPA, 0.91 +/- 0.45 x 10(-2) pmol NE, and 0.290 +/- 0.087 pmol DA (mean values per microg total protein, n = 4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-DL-m-tyrosine (alpha-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K+. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1-10 microM. In juveniles induced by 10 microM DDTC, levels of both NE and DA averaged approximately 80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.     

Anti-inflammatory and neuroprotective effects of magnolol in chemical hypoxia in rat cultured cortical cells in hypoglycemic media

Our previous studies demonstrated that magnolol protects neurons against chemical hypoxia by KCN in cortical neuron-astrocyte mixed cultures (14). In the present study, we examined whether the neuroprotective effect of magnolol involve modulating inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), induced by KCN (hypoxia) or KCN plus lipopolysaccharide (LPS). In glucose-absent (hypoglycemia) media, KCN or KCN plus LPS induced increases in lactate dehydrogenase (LDH) activity by 32% and 34%, and PGE2 production by 12% and 32%, respectively. Both LDH and PGE2 increases were suppressed by 100 microM magnolol. In addition, although KCN or LPS alone did not increase NO generation, KCN plus LPS increased NO generation. This increase was reduced by 100 microM magnolol or 10 microM L-NAME, but the LDH increase and PGE2 production were not reduced by L-NAME. These findings suggest that the protective effects of magnolol against brain damage by KCN or KCN plus LPS in hypoglycemic media may involve inhibition of PGE2 production, but inhibition of NO generation may not be important.     

Effects of PKC isozyme inhibitors on constrictor responses in the feline pulmonary vascular bed

The effects of G?-6976, a Ca(2+)-dependent protein kinase C (PKC) isozyme inhibitor, and rottlerin, a PKC-delta isozyme/calmodulin (CaM)-dependent kinase III inhibitor, on responses to vasopressor agents were investigated in the feline pulmonary vascular bed. Injections of angiotensin II, norepinephrine (NE), serotonin, BAY K 8644, and U-46619 into the lobar arterial constant blood flow perfusion circuit caused increases in pressure. G?-6976 reduced responses to angiotensin II; however, it did not alter responses to serotonin, NE, or U-46619, whereas G?-6976 enhanced BAY K 8644 responses. Rottlerin reduced responses to angiotensin II and NE, did not alter responses to serotonin or U-46619, and enhanced responses to BAY K 8644. Immunohistochemistry of feline pulmonary arterial smooth muscle cells demonstrated localization of PKC-alpha and -delta isozymes in response to phorbol 12-myristate 13-acetate and angiotensin II. Localization of PKC-alpha and -delta isozymes decreased with administration of G?-6976 and rottlerin, respectively. These data suggest that activation of Ca(2+)-dependent PKC isozymes and Ca(2+)-independent PKC-delta isozyme/CaM-dependent kinase III mediate angiotensin II responses. These data further suggest that Ca(2+)-independent PKC-delta isozyme/CaM-dependent kinase III mediate responses to NE. A rottlerin- or G?-6976-sensitive mechanism is not involved in mediating responses to serotonin and U-46619, but these PKC isozyme inhibitors enhanced BAY K 8644 responses in the feline pulmonary vascular bed.     

Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.     

Effect of hypoxia and glucose deprivation on ATP level, adenylate energy charge and [Ca]0-dependent and independent release of [H]dopamine in rat striatal slices

Release of [³H]dopamine ([³H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [³H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca²⁺]₀-independent release of [³H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 ± 4.15 to 6.98 ± 1.83 mol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C. under glucose-free conditions was significantly reduced from 0.73 ± 0.04 to 0.44 ± 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 ± 0.11 mol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 ± 0.37 nmol/mg protein. The resting release was enhanced in a [Ca²⁺]₀-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 μM ouabain, inhibitor of Na⁺/K⁺-activated ATPase, enhanced the release of [³H]DA at rest in a [Ca²⁺]₀-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na⁺/K⁺-ATPase that results in reversal of the uptake processes and in [Ca²⁺]₀-independent [³H]DA release from the axon terminals.