Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger

Na(+) cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na(+)/Ca(2+) exchanger, bumetanide for the Na(+)-K(+)-2Cl(-) cotransporter, and amiloride for Na(+)/H(+) cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na(+)/Ca(2+) exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na(+)/H(+) cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na(+)-K(+)-2Cl(-) cotransporter and Na(+)/Ca(2+) exchanger functions are key to cerebral vascular oxygen responses.     

Apple procyanidins induce hyperpolarization of rat aorta endothelial cells via activation of K+ channels

Apple procyanidins (AP), one of the polyphenol-rich compounds, showed an endothelial-dependent vasorelaxation in rat aorta, but the mechanisms of beneficial effects are still unclear. The present study was designed to clarify the potential role of AP in rat aorta endothelial cells (RAECs). The treatment of RAECs with AP (1-10 μg/ml) resulted in a dose-dependent hyperpolarization with a maximum effect at 10 μg/ml, and for this reason, AP (10 μg/ml) was used in all the following experiments. AP-induced hyperpolarization was significantly inhibited by pretreatment of nonspecific K(+) inhibitor, tetraethyl ammonium chloride or specific K(+) channel inhibitors, iberiotoxin, glibenclamide, 4-aminopyridine and BaCl(2), as well as by high KCl or Ca(2+)-free solution. AP-induced hyperpolarization was also proved using 64-channel multielectrode dish system that can monitor a direct and real-time change of membrane potential. Furthermore, AP treatment caused a significant increase of nitric oxide (NO) production and cyclic guanosine monophosphate levels via endothelial NO synthase messenger RNA expression. The NO production was inhibited by N(G)-monoethyl-l-arginine or Ca(2+)-free solution and was completely abolished by their combination. Also, AP inhibited endothelial proliferation, while the effect was significantly abolished by N(G)-monoethyl-l-arginine or tetraethyl ammonium chloride. These findings suggest that AP induces both hyperpolarization of RAECs via multiple activation of K(+) channels and activation of NO/cyclic guanosine monophosphate pathway via increasing NO production or is responsible for antiangiogenic effect. Diminishment of hyperpolarization as well as NO production of AP in Ca(2+)-free solution implicated that AP would play a crucial role in promoting Ca(2+) influx into endothelial cells so as to promote both actions.     

Evidence of calcium- and SNARE-dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization

The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH(3) cells that express voltage-dependent calcium channels and are subjected to Ca(2+) oscillations, we found that treatment with high K(+)-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH(3) cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH(3) cells and rat brain synaptosomes showed that K(+)-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH(3) cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex.     

Calcium-independent phospholipase A2 in rat tissue cytosols

Cytosols (105,000 X g supernatant) from seven rat tissues were assayed for Ca2+-independent phospholipase A2 activity with either 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 pmol/min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca2+-independent phospholipase A2 originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca2+-dependent phospholipase A2 activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca2+-dependent phospholipase A2 activity, however, came from lung cells. Neither this enzyme nor the Ca2+-independent phospholipase A2 from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca2+-dependent phospholipase A2 isolated from rat liver mitochondria.     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.     

Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor) or amiloride (Na(+)/H(+) exchange channel inhibitor). Suppressing amiloride-sensitive Na(+)/H(+) exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na(+)-K(+)-2Cl(-) channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na(+)/Ca(2+) exchanger extrudes Na(+) in exchange for Ca(2+), thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na(+)/Ca(2+) exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na(+)-K(+)-2Cl(-) channels. The Na(+)/Ca(2+) exchanger then extrudes Na(+) and increases endothelial Ca(2+). The increase in endothelial Ca(2+) causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.     

Inhibitory effect of calcium-binding protein regucalcin on Ca2(+)-activated DNA fragmentation in rat liver nuclei

Incubation of isolated rat liver nuclei with ATP, NAD+, and micromolar Ca2+ concentrations of various metal ions resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 1.0 microM Ca2+ added, and saturation of the process was observed with 10 microM Ca2+. The Ca2+ (10 microM)-activated DNA fragmentation was inhibited by the presence of Ca2(+)-binding protein regucalcin isolated from rat liver cytosol. The inhibitory effect of regucalcin was complete at 0.5 microM. At 25 microM Ca2+ added, such an effect of regucalcin (1.0 microM) was not seen. Regucalcin also inhibited Ca2(+)-activated DNA fragmentation in the presence of calmodulin (10 and 20 micrograms). The results show that regucalcin can inhibit the Ca2(+)-activated DNA fragmentation due to binding the metal, suggesting a role in regulation of liver nuclear functions.     

Mechanism of cGMP contribution to the vasodilator response to NO in rat middle cerebral arteries

This study examined the mechanism by which cGMP contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries (MCA). Administration of a NO donor, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate), or 8-bromo-cGMP (8-BrcGMP) increased the diameter of serotonin-preconstricted MCA by 79 +/- 3%. The response to DEA-NONOate, but not 8-BrcGMP, was attenuated by iberiotoxin (10(-7) M) or a 80 mM high-K(+) media, suggesting that activation of K(+) channels contributes to the vasodilator response to NO but not 8-BrcGMP. The effects of NO and cGMP on the vasoconstrictor response to Ca(2+) were also studied in MCA that were permeabilized with alpha-toxin and ionomycin. Elevations in bath Ca(2+) from 10(-8) to 10(-5) M decreased the diameter of permeabilized MCA by 76 +/- 5%. DEA-NONOate (10(-6) M) and 8-BrcGMP (10(-4) M) blunted this response by 60%. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (10(-5) M) blocked the inhibitory effect of the NO donor, but not 8-BrcGMP, on Ca(2+)-induced vasoconstriction. 8-BrcGMP (10(-4) M) had no effect on intracellular Ca(2+) concentration ([Ca(2+)](i)) in control, serotonin-stimulated, or alpha-toxin- and ionomycin-permeabilized vascular smooth muscle cells isolated from the MCA. These results indicate that the vasodilator response to NO in rat MCA is mediated by activation of Ca(2+)-activated K(+) channels via a cGMP-independent pathway and that cGMP also contributes to the vasodilator response to NO by decreasing the contractile response to elevations in [Ca(2+)](i).     

Inhibition of K(Ca) channels restores blunted hypoxic pulmonary vasoconstriction in rats with cirrhosis

Rats with liver cirrhosis exhibit the hepatopulmonary syndrome composed of blunted hypoxic pulmonary vasoconstriction and arterial hypoxemia. The purpose of this study was to investigate the roles of nitric oxide (NO) and endothelin-1 (ET-1) in the blunted hypoxic pressor response (HPR) in rats with common bile duct ligation (CBDL). Lungs from CBDL rats exhibited markedly blunted HPR, increased endothelial NO synthase (NOS) protein expression, and decreased ET-1 mRNA and peptide expression. The blunted HPR was not reversed by sequential NOS and soluble guanylyl cyclase inhibition by nitro-L-arginine and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), respectively, or by NOS inhibition combined with ET-1 addition. The blunted HPR was not due to a generalized inability to vasoconstrict because perfusion pressure was equally elevated by increased perfusate KCl in CBDL and sham lungs. After KCl vasoconstriction, HPR was potentiated and did not differ between CBDL and sham lungs. Blunted HPR was also completely restored in CBDL lungs treated with nitro-L-arginine, ODQ, and the Ca(2+)-activated K(+) channel blockers apamin and charybdotoxin. These results indicate that although CBDL-induced liver cirrhosis is associated with increased NO and decreased ET-1 in the lung, the blunted HPR is a result of additional factors and appears to involve Ca(2+)-activated K(+) channel activation.     

Polyclonal antibody against an inducible form of nitric oxide synthase purified from the liver of rats treated with Propionibacterium acnes and lipopolysaccharide.

A polyclonal antibody was raised in the rabbit against an inducible form of nitric oxide (NO) synthase (EC 1.14.23) purified from the liver of rats with acute liver necrosis induced by i.v. administration of Propionibacterium acnes and lipopolysaccharide. The antibody immunoprecipitated NO synthase activities in the soluble extract of the liver from treated rats. Western blot analysis showed that the cytosols of the liver, lung and spleen from the treated rats but not from non-treated rats, and that of murine macrophages cultured in the presence of lipopolysaccharide and interferon-gamma, contained immunoreactive protein with a molecular weight of 125 kDa. The antibody, however, does not cross-react with a 150 kDa constitutive form of NO synthase present in the brain of rats, indicating that the inducible and constitutive enzymes are immunologically distinguishable.     

Electrophysiological characterization and ionic stoichiometry of the rat brain K(+)-dependent NA(+)/CA(2+) exchanger, NCKX2

We have recently described a novel K(+)-dependent Na(+)/Ca(2+) exchanger, NCKX2, that is abundantly expressed in brain neurons (Tsoi, M., Rhee, K.-H., Bungard, D., Li, X.-F., Lee, S.-L., Auer, R. N., and Lytton, J. (1998) J. Biol. Chem. 273, 4115--4162). The precise role for NCKX2 in neuronal Ca(2+) homeostasis is not yet clearly understood but will depend upon the functional properties of the molecule. Here, we have performed whole-cell patch clamp analysis to characterize cation dependences and ion stoichiometry for rat brain NCKX2, heterologously expressed in HEK293 cells. Outward currents generated by reverse NCKX2 exchange depended on external Ca(2+) with a K(12) of 1.4 or 101 microm without or with 1 mm Mg(2+), and on external K(+) with a K(1/2) of about 12 or 36 mm with choline or Li(+) as counter ion, respectively. Na(+) inhibited outward currents with a K(1/2) of about 60 mm. Inward currents generated by forward NCKX2 exchange depended upon external Na(+) with a K(1/2) of 30 mm and a Hill coefficient of 2.8. K(+) inhibited the inward currents by a maximum of 40%, with a K(1/2) of 2 mm or less, depending upon the conditions. The transport stoichiometry of NCKX2 was determined by observing the change in reversal potential as individual ion gradients were altered. Our data support a stoichiometry for rat brain NCKX2 of 4 Na(+):(1 Ca(2+) + 1 K(+)). These findings provide the first electrophysiological characterization of rat brain NCKX2, and the first evidence that a single recombinantly expressed NCKX polypeptide encodes a K(+)-transporting Na(+)/Ca(2+) exchanger with a transport stoichiometry of 4 Na(+):(1 Ca(2+) + 1 K(+)).     

Vasorelaxant effects of macrocyclic bis(bibenzyls) from liverworts

Vasorelaxant effects of a series of bis(bibenzyls) from liverworts such as Marchantia polymorpha and Marchantia paleacea on rat aorta demonstrated that they relaxed phenylephrine (PE)-induced contractions, which may be mediated through the increased release of NO from endothelial cells as well as opening of K(+) channels, and inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs). Structure-activity relationship based on their structures was discussed. The presence of two aromatic rings which can be connected through two atoms bridge spacer may play an important role for vasorelaxant effect.     

Characterizing voltage-dependent Ca(2+) channels coupled to VIP release and NO synthesis in enteric synaptosomes

In enteric synaptosomes of the rat, the role of voltage-dependent Ca(2+) channels in K(+)-induced VIP release and nitric oxide (NO) synthesis was investigated. Basal VIP release was 39 +/- 4 pg/mg, and cofactor-substituted NO synthase activity was 7.0 +/- 0.8 fmol. mg(-1). min(-1). K(+) depolarization (65 mM) stimulated VIP release Ca(2+) dependently (basal, 100%; K(+), 172.2 +/- 16.2%; P < 0.05, n = 5). K(+)-stimulated VIP release was reduced by blockers of the P-type (omega-agatoxin-IVA, 3 x 10(-8) M) and N-type (omega-conotoxin-GVIA, 10(-6) M) Ca(2+) channels by ~50 and 25%, respectively, but not by blockers of the L-type (isradipine, 10(-8) M), Q-type (omega-conotoxin-MVIIC, 10(-6) M), or T-type (Ni(2+), 10(-6) M) Ca(2+) channels. In contrast, NO synthesis was suppressed by omega-agatoxin-IVA, omega-conotoxin-GVIA, and isradipine by ~79, 70, and 70%, respectively, whereas Ni(2+) and omega-conotoxin-MVIIC had no effect. These findings are suggestive of a coupling of depolarization-induced VIP release primarily to the P- and N-type Ca(2+) channels, whereas NO synthesis is presumably dependent on Ca(2+) influx not only via the P- and N- but also via the L-type Ca(2+) channel. In contrast, none of the Ca(2+) channel blockers affected VIP release evoked by exogenous NO, suggesting that NO induces VIP secretion by a different mechanism, presumably involving intracellular Ca(2+) stores.     

Gamma-irradiation potentiates L-arginine-dependent nitric oxide formation in mice.

Gamma-irradiation of mongrel mice at a sublethal dose (700 Roentgen) enhanced the formation of nitric oxide (NO) in the liver, intestine, lung, kidney, brain, spleen or heart of the animals. NO formation was determined by the increase in intensity of the EPR signal due to trapping of NO into mononitrosyl iron complexes (MNIC) with exogenous diethyldithiocarbamate (DETC) injected intraperitoneally. The EPR signal of these MNIC-DETC complexes was characterized by g-factor values at g perpendicular values at g perpendicular = 2.035 and g parallel = 2.02 and a triplet hyperfine structure at g perpendicular. The NO synthase inhibitor, NG-nitro-L-arginine, prevented MNIC-DETC complex formation both in liver and intestine, demonstrating the involvement of endogenous NO formed. Thus, gamma-irradiation may enhance endogenous NO biosynthesis in these tissues, presumably by facilitating the entry of Ca2+ ions into the membrane as well as the cytosol of NO-producing cells through irradiation-induced membrane lesions.     

Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain.

In a search for the non-muscle equivalent of calsequestrin (the low-affinity high-capacity Ca2(+)-binding protein responsible for Ca2+ storage within the terminal cisternae of the sarcoplasmic reticulum), acidic proteins were extracted from rat liver and brain microsomal preparations and purified by column chromatography. No calsequestrin was observed in these extracts, but the N-terminal amino acid sequence of the major Ca2(+)-binding protein of the liver microsomal fraction was determined and found to correspond to that of calreticulin. This protein was found to bind approx. 50 mol of Ca2+/mol of protein, with low affinity (average Kd approx. 1.0 mM). A monoclonal antibody, C6, raised against skeletal-muscle calsequestrin cross-reacted with calreticulin in SDS/PAGE immunoblots, but polyclonal antibodies reacted with native calreticulin only weakly, or not at all, after SDS denaturation. Immuno-gold decoration of liver ultrathin cryosections with affinity-purified antibodies against liver calreticulin revealed luminal labelling of vacuolar profiles indistinguishable from calciosomes, the subcellular structures previously identified by the use of anti-calsequestrin antibodies. We conclude that calreticulin is the Ca2(+)-binding protein segregated within the calciosome lumen, previously described as being calsequestrin-like. Because of its properties and intraluminal location, calreticulin might play a critical role in Ca2+ storage and release in non-muscle cells, similar to that played by calsequestrin in the muscle sarcoplasmic reticulum.     

A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.     

Rat brain endoplasmic reticulum calcium pools are anatomically and functionally segregated.

Autoradiographic imaging can localize 45Ca2+ selectively accumulated via the Ca2+, Mg2(+)-ATPase into endoplasmic reticulum stores in rat brain slices. 45Ca2+ accumulation is markedly stimulated by oxalate and displays a heterogeneous distribution which resembles the mRNA distribution for a sarcoendoplasmic reticulum Ca2+, Mg2(+)-ATPase. Inositol 1,4,5-triphosphate [I(1,4,5)P3] inhibits 45Ca2+ accumulation selectively into regions corresponding to those enriched in I(1,4,5)P3 receptor-binding sites and in Ca2+, -Mg2(+)-ATPase mRNA. Thus rat brain endoplasmic reticulum calcium stores are anatomically and functionally differentiated.     

Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin.

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca2(+)-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.