Contribution of peroxynitrite to fatal cardiovascular depression induced by overproduction of nitric oxide in rostral ventrolateral medulla of the rat

We evaluated the contribution of peroxynitrite to the fatal cardiovascular depression induced by overproduction of nitric oxide (NO) after activation of inducible NO synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of E. coli lipopolysaccharide (LPS) bilaterally into the RVLM elicited significant hypotension, bradycardia, reduction in sympathetic vasomotor tone and mortality. There was also a discernible elevation of iNOS expression in the ventrolateral medulla, followed by a massive production of nitrotyrosine, an experimental index for peroxynitrite. Co-administration bilaterally into the RVLM of the selective iNOS inhibitor, S-methylisothiourea (50, 100 or 250 pmol), an active peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis- (N-methyl-4'-pyridyl)-porphyrinato iron (III) (10 or 50 pmol), a peroxynitrite scavenger, L-cysteine (5, 50 or 100 pmol), or a superoxide dismutase mimetic, Mn(III)-tetrakis-(4-benzoic acid) porphyrin (1 or 10 pmol), significantly prevented mortality, reduced nitrotyrosine production and reversed the NO-induced cardiovascular suppression after application of LPS into the RVLM. We conclude that the formation of peroxynitrite by a reaction between superoxide anion and NO is primarily responsible for the fatal cardiovascular depression induced by overproduction of NO after activation of iNOS at the RVLM.     

Increased expression of VDAC1 sensitizes carcinoma cells to apoptosis induced by DNA cross-linking agents

A major clinical problem regarding antitumoral treatment with DNA cross-linking agents such as cisplatin (Cisp), mechlorethamine (HN2) or its derivative melphalan (MLP) is intrinsic or acquired resistance to therapy, which frequently results from a resistance to apoptosis induction. In this study, aimed to identify novel sensitizing targets to DNA cross-linker-induced cell death, we demonstrated that MLP, Cisp and HN2 induce mitochondrial permeability transition pore (PTP)-mediated apoptosis in cervical and colon carcinoma cells. This apoptotic pathway is characterized by dissipation of the mitochondrial membrane potential, production of ROS, mitochondrial translocation of Bax, release of apoptogenic factors, caspase activation and nuclear alterations. The opening of PTP and subsequent apoptosis was reduced in Bax deficient cells and in cells with elevated Bcl-2 level, but not in cells invalidated for Bak. We further showed that, among the pro-apoptotic PTP regulators tested (VDAC1, creatine kinase, ANT1 and ANT3), exogenous overexpression of VDAC1 was the most effective in enhancing Cisp- and MLP-induced apoptosis. In addition, pharmacologically induced up-regulation of VDAC1 by the chemotherapeutic agent arsenic trioxide (As(2)O(3)) greatly sensitized HeLa cells to Cisp and MLP treatment. These data indicate that increased expression of VDAC1 appears as a promising strategy to improve DNA cross-linker-induced chemotherapy.     

Differential engagements of glutamate and GABA receptors in cardiovascular actions of endogenous nNOS or iNOS at rostral ventrolateral medulla of rats

1. We evaluated in Sprague-Dawley rats anaesthetized with propofol the engagement of soluble guanylyl cyclase (sGC)/cGMP cascade, glutamatergic and GABAergic neurotransmission in the cardiovascular actions of endogenous nitric oxide (NO) at the rostral ventrolateral medulla (RVLM). 2. Microinjection bilaterally into the RVLM of a selective iNOS inhibitor, S-methylisothiourea (SMT, 250 pmoles), or a selective nNOS inhibitor, 7-nitroindazole (7-NI, 5 pmoles), induced respectively an enhancement or a reduction in systemic arterial pressure, heart rate and power density of the vasomotor components in the spectrum of arterial blood pressure signals, our experimental index for sympathetic neurogenic vasomotor tone. 3. The cardiovascular actions of SMT or 7-NI in the RVLM were significantly antagonized by co-administration into the RVLM of the sGC inhibitor, 1H-[1,2,4]Oxadiazole[4,3-alpha]quinoxalin-1-one (ODQ, 250 or 500 pmoles). 4. The cardiovascular excitatory effects after blockade of endogenous iNOS activity were significantly attenuated when N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (20 or 50 pmoles), or non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (250 or 500 pmoles), was co-microinjected bilaterally into the RVLM. 5. On the other hand, the cardiovascular depressive responses to blockade of endogenous nNOS activity were significantly antagonized on co-administration of GABA(A) receptor antagonist, bicuculline methiodine (5 or 10 pmoles), but not GABA(B) receptor antagonist, 2-hydroxy saclofen (50 or 100 pmoles). 6. We conclude that the cardiovascular actions of endogenous NO in the RVLM engage the sGC/cGMP pathway. In addition, whereas NO derived from nNOS induced sympathoexcitation via both NMDA and non-NMDA receptors in the RVLM, NO generated by iNOS elicited sympathoinhibition via GABA(A) receptors.     

Mitochondrial respiratory enzyme complexes in rostral ventrolateral medulla as cellular targets of nitric oxide and superoxide interaction in the antagonism of antihypertensive action of eNOS transgene

Overproduction of nitric oxide (NO) by gene transduction of endothelial NO synthase (eNOS) in rostral ventrolateral medulla (RVLM), which is responsible for maintenance of vasomotor tone, reduces arterial pressure in spontaneously hypertensive rats (SHR). This NO-induced vasodepression, however, is not sustained and is followed by rebound hypertension. Because superoxide anion (O(2)(*-)) level is increased and synthesis or activity of mitochondrial manganese superoxide dismutase (SOD2) is reduced in RVLM during hypertension, we hypothesized that an interaction between NO and O(2)(*-) in RVLM, using mitochondrial respiratory enzyme complexes (MRC) as the cellular target, contributes to those cardiovascular outcomes after eNOS gene transduction in SHR. The present study assessed this hypothesis using adenoviral vectors to overexpress eNOS (AdeNOS) and/or SOD2 (AdSOD2) in RVLM of SHR or normotensive Wistar-Kyoto (WKY) rats. Microinjection of AdeNOS bilaterally into RVLM elicited 35% depression of MRC-I enzyme activity and evoked 60% and 50% increase in O(2)(*-) and peroxynitrite level in RVLM of SHR, but not WKY rats, which was reversed by cotransduced AdSOD2 or treatment with peroxynitrite decomposition catalyst. Cotransduction of AdeNOS and AdSOD2 in RVLM of SHR elicited significantly greater decreases in arterial pressure and heart rate than those promoted by the individual transgene and prevented the AdeNOS-induced rebound hypertension. We conclude that an interactive action between NO and O(2)(*-) on MRC-I in RVLM via formation of peroxynitrite contributes to the unsustained hypotensive effects of NO after overexpression of eNOS in SHR. The mitochondria-derived O(2)(*-) also mediates the rebound hypertension induced by eNOS transgene in RVLM of SHR.     

Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat

The organophosphate poison mevinphos (Mev) elicits cardiovascular responses via nitric oxide (NO) produced on activation of M2 muscarinic receptors (M2R) in the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates. This study further evaluated the contribution of nitric oxide synthase (NOS) isoforms at the RVLM to this process, using adult Sprague-Dawley rats. Bilateral co-microinjection into the RVLM of the selective NOS I inhibitor (250 pmol), 7-nitroindazole or N(omega)-propyl-L-arginine antagonized the initial sympathoexcitatory cardiovascular responses to Mev (10 nmol). Co-administration of a selective NOS II inhibitor, N6-(1-iminoethyl)-L-lysine (250 or 500 pmol) further enhanced these cardiovascular responses and reversed the secondary sympathoinhibitory actions of Mev. A potent NOS III inhibitor, N5-(1-iminoethyl)-L-ornithine (46 or 92 nmol) was ineffective. We also found that M2R co-localized only with NOS I- or NOS II-immunoreactive RVLM neurons. Furthermore, only NOS I or II in the ventrolateral medulla exhibited an elevation in mRNA or protein levels during the sympathoexcitatory phase, with further up-regulated synthesis of NOS II during the sympathoinhibitory phase of Mev intoxication. We conclude that whereas NOS III is not engaged, NO produced by NOS I and II in the RVLM plays, respectively, a sympathoexcitatory and sympathoinhibitory role in the cardiovascular responses during Mev intoxication.     

Nitric oxide modulates cardiovascular function in the rat by activating adenosine A2A receptors and inhibiting acetylcholine release in the rostral ventrolateral medulla

1. Nitric oxide (NO), a gas transmitter, modulates many physiological processes, including the central regulation of cardiovascular activity. However, the mechanisms underlying the regulation of cardiovascular activity remain relatively unexplored. In the present study, we hypothesized that central NO-dependent sympathetic inhibition is mediated by activation of adenosine A(2A) receptors (A(2A)R) and inhibition of acetylcholine (ACh) release in the rostral ventrolateral medulla (RVLM). 2. L-Arginine (L-Arg; an NO donor; 100 nmol/100 nL) was microinjected into the RVLM of male Sprague-Dawley rats and heart rate variability (HRV) was assessed as an index of cardiac sympathovagal balance. Following microinjection of L-Arg, decreases were seen in mean arterial pressure (MAP), heart rate (HR) and the ratio of the low- to high-frequency components (LF/HF) of HRV. Pretreatment of rats with SCH58261 (40 pmol/60 nL into the RVLM), a competitive antagonist of the A(2A) R, attenuated these effects. 3. Western blot analysis and ELISA revealed that adenosine and A(2A)R levels increased in the RVLM following L-Arg microinjection, whereas ACh and muscarinic M(1) receptor levels decreased significantly, in parallel with the cardiovascular responses to L-Arg microinjection. The decrease in ACh levels was abolished by SCH58261 pretreatment. 4. Microinjection of N(G)-nitro-L-arginine methyl ester (a non-selective inhibitor of NO synthase; 15 nmol/100 nL) into the RVLM significantly increased MAP, HR and sympathetic activity, as evidenced by HRV (LF, HF and the LF/HF ratio were all increased). 5. The results indicate that the central NO/NO synthase system in the RVLM may modulate cardiovascular activity by activating the A(2A)R, which subsequently inhibits activation of the muscarinic M(1) receptor.     

Phasic cardiovascular responses to mevinphos are mediated through differential activation of cGMP/PKG cascade and peroxynitrite via nitric oxide generated in the rat rostral ventrolateral medulla by NOS I and II isoforms

The organophosphate insecticide mevinphos (Mev) acts on the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates, to elicit phasic cardiovascular responses via nitric oxide (NO) generated by NO synthase (NOS) I and II. We evaluated the contribution of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade and peroxynitrite in this process. PKG expression in ventrolateral medulla of Sprague-Dawley rats manifested an increase during the sympathoexcitatory phase (Phase I) of cardiovascular responses induced by microinjection of Mev bilaterally into the RVLM that was antagonized by co-administration of 7-nitroindazole or Nomega-propyl-L-arginine, two selective NOS I inhibitors or 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (ODQ), a selective sGC antagonist. Co-microinjection of ODQ or two PKG inhibitors, KT5823 or Rp-8-Br-cGMPS, also blunted the Mev-elicited sympathoexcitatory effects. However, the increase in nitrotyrosine, a marker for peroxynitrite, and the sympathoinhibitory circulatory actions during Phase II Mev intoxication were antagonized by co-administration of S-methylisothiourea, a selective NOS II inhibitor, Mn(III)-tetrakis-(4-benzoic acid) porphyrin, a superoxide dismutase mimetic, 5,10,15,20-tetrakis-N-methyl-4'-pyridyl)-porphyrinato iron (III), a peroxynitrite decomposition catalyst, or L-cysteine, a peroxynitrite scavenger. We conclude that sGC/cGMP/PKG cascade and peroxynitrite formation may participate in Mev-induced phasic cardiovascular responses as signals downstream to NO generated respectively by NOS I and II in the RVLM.     

Farnesyltransferase inhibitor BMS-214662 induces apoptosis in myeloma cells through PUMA up-regulation, Bax and Bak activation, and Mcl-1 elimination

We have studied the mechanism of apoptosis elicited by the farnesyltransferase inhibitor (R)-7-cyano-2,3,4,5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine (BMS-214662) in human myeloma cell lines. Low concentrations of BMS-214662 efficiently inhibited protein farnesylation but did not affect the activation of Akt. BMS-214662 treatment increased levels of the BH3-only protein PUMA; induced proapoptotic conformational changes of Bax and Bak; reduced Mcl-1 levels; caused mitochondrial transmembrane potential loss; induced cytochrome c release, caspase activation, apoptosis-inducing factor (AIF) nuclear translocation, and phosphatidylserine exposure; and allowed the development of apoptotic morphology. Western blot analysis of cell extracts revealed the activation of caspases 2, 3, 8, and 9 upon treatment with BMS-214662. The general caspase inhibitor Z-VAD-fmk significantly prevented BMS-214662-induced death in U266 and RPMI 8226 cells but not in NCI-H929 cells. A mixture of selective caspase inhibitors for caspases 9 [N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone (Z-LEHD-fmk)], 3 (Z-DEVD-fmk), and 6 (Z-VEID-fmk) approached the protective effect of Z-VAD upon cell death. However, Z-VAD-fmk did not prevent BMS-214662-induced Bax and Bak activation and decrease of Mcl-1 levels. According to its effect on cell death, Z-VAD-fmk inhibited nuclear translocation of AIF in RPMI 8226 and U266 but not in NCI-H929 cells. These results suggest that apoptosis triggered by BMS-214662 is initiated by a PUMA/Bax/Bak/Mcl-1-dependent mechanism. In some cell lines, Bax/Bak activation is not sufficient per se to induce mitochondrial failure and release of apoptogenic proteins, and so caspases need to be activated to facilitate apoptosis. After DeltaPsi(m) loss, execution of apoptosis was performed in all cases by a cytochrome c-enabled, caspase-9-triggered, caspase cascade and the nuclear action of AIF.     

Role of nitric oxide on pressor mechanisms within the dorsomedial and rostral ventrolateral medulla in anaesthetized cats

1. The role of nitric oxide (NO) in central cardiovascular regulation and the correlation between NO and glutamate-induced mechanisms is not clear. Microinjection of glutamate (3 nmol/30 nL) into dorsomedial medulla (DM) and rostral ventrolateral medulla (RVLM) increased arterial blood pressure (BP) and sympathetic vertebral nerve activity (VNA). Thus, in the present study, we examined the modulation by NO of glutamate-induced pressor responses in the DM and RVLM of cats. 2. Histochemical methods using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) as a marker to stain neurons containing NO synthase (NOS), showed positive findings of NOS in both the DM and RVLM. 3. Microinjection of N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, into the DM or RVLM did not alter resting BP and VNA, but it did cause a dose-dependent attenuation of glutamate-induced pressor responses. Interestingly, the increase in NO levels that resulted from pretreatment with L-arginine (L-Arg) or sodium nitroprusside (SNP) did not alter resting BP and VNA, but still inhibited glutamate-induced pressor responses in the DM and RVLM in a dose-dependent manner. 4. We also examined whether NO modulated the pressor responses induced by activation of different excitatory amino acid receptors. N-Methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) were used. Consistent with the results from the initial glutamate studies, we observed that not only L-NAME, but also L-Arg and SNP attenuated pressor responses induced by NMDA and AMPA. No difference was found between the effects of NO on NMDA- and AMPA-induced pressor responses. 5. To investigate the possibility of a loss of agonist selectivity, the effects of D-2-amino-5-phosphonovalerate (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on AMPA and NMDA responses in the DM were examined. The results showed that CNQX did not alter NMDA-induced pressor responses, while D-AP5 failed to alter AMPA-induced responses. 6. Our results suggest that activation of the glutamate-induced pressor mechanism is regulated by changes in NO levels in the DM and RVLM. This implies that NO may play a permissive role to allow operation of the glutamate-activation mechanism.     

CONTROL OF SYMPATHETIC OUTFLOWS BY THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS

1. The functional role of the paraventricular nucleus (PVN) has been examined by studying its connections with cardiovascular neurons in the medulla and spinal cord and its influence on activity in several sympathetic nerves. 2. Chemical stimulation of neurons within the PVN can elicit pressor responses and can excite reticulo-spinal vasomotor neurons in the rostral ventrolateral medulla (RVLM). 3. The PVN-RVLM excitation is blocked by kynurenic acid applied iontophoretically in the vicinity of RVLM-spinal neurons, suggesting this is a glutamate-dependent pathway. 4. Electrical stimulation of PVN neurons evoked action potentials in RVLM neurons after 27 ms with a small variability. 5. Anterograde and retrograde labelling of PVN and RVLM neurons revealed PVN terminals closely associated with RVLM-spinal neurons and showed that the PVN is connected to the spinal cord via three pathways. 6. Chemical activation of PVN neurons can produce a pattern of activation of cardiovascular neurons similar to that occurring in defence against plasma volume expansion. 7. It is concluded that the PVN connections with the RVLM and spinal cord are important to a role in defending against life-threatening disturbances.     

cGMP/protein kinase G-dependent potentiation of glutamatergic transmission induced by nitric oxide in immature rat rostral ventrolateral medulla neurons in vitro

Although both nitric oxide (NO) and glutamate within the rostral ventrolateral medulla (RVLM) are important mediators of the central cardiovascular regulation, little is known about the functional interactions between these two mediators. Herein, we investigated the possible role of NO on the glutamatergic transmission of RVLM neurons. Whole-cell patch-clamp recordings were performed on visualized RVLM neurons in the brainstem slice preparation of rats. We found that bath application of l-arginine, the substrate for NO production, significantly increased the amplitude of excitatory postsynaptic currents (EPSCs). This enhancement was completely abolished by coadministration of the NO synthase inhibitor 7-nitroindazole and mimicked by the NO donors 3-morpholinylsydnoneimine and spermine NONOate. Bath application of a NO-sensitive guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or a protein kinase G (PKG) inhibitor, Rp-8-bromo-guanosine 3',5'-cyclic monophosphorothioate, fully prevented the l-arginine-, 3-morpholinylsydnoneimine-, and N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl]-1,3-propanediamin (spermine NONOate)-induced synaptic potentiation. Direct activation of PKG with 8-(4-chlorophenylthio)-cGMP mimicked the action of NO donors. Furthermore, the augmentation by spermine NONOate of EPSC was accompanied by a reduction of the paired-pulse facilitation and synaptic failure rate of EPSCs. Spermine NONOate also significantly increased the frequency of both spontaneous and miniature EPSCs without altering their amplitude distribution. Pretreatment with the N-type Ca2+ channel blocker omega-conotoxin GVIA selectively blocked the spermine NONOate-induced synaptic potentiation. These results suggest that NO acts presynaptically to elicit a synaptic potentiation on the RVLM neurons through an enhancement of presynaptic N-type Ca2+ channel activity leading to facilitating glutamate release. The presynaptic action of NO is mediated by a cGMP/PKG-coupled signaling pathway.     

RU486对硫酸皮质酮引起的头端延髓腹外侧区心血管神经元作用的影响

目的研究糖皮质激素（ＧＣ）的非基因组机制在交感神经活动调节中的作用。方法细胞外记录氨基甲酸乙酯麻醉的ＳＤ大鼠头端延髓腹外侧区（ＲＶＬＭ）被鉴定的心血管神经元的自发放电,观察微电泳硫酸皮质酮（Ｏ３ＲＴ对心血管神经元活动的影响,及其 ＧＣ受体拮抗剂 ＲＵ ４８６对ＣＯＲＴ导致神经元作用的影响。结果在ＲＶＬＭ共记录到３３个心血管神经元,微电泳硫酸皮质酮（ＣＯＲＴ）后２５（７６％）个神经元放电迅速加快,且具有剂量依赖性,余８个（２７％）心血管神经元自发放电没有变化;其中被ＣＯＲＴ兴奋的１２个单位微电泳ＲＵ４８６（糖皮质激素受体阻断剂）后神经元的基础放电没有明显变化（Ｐ＞０．０５）,但能部分（９个单位）或完全（３个单位）阻断硫酸皮质酮导致的兴奋作用。结论Ｏ３ＲＴ对ＲＶＬＭ心血管神经元具有快速的兴奋作用,ＲＵ４８６并不改变ＲＶＬＭ心血管神经元的基础活动,但能部分或完全阻断ＣＯＲＴ导致的神经元兴奋作用。     

Identification of PKC isozymes and effect of knockdown of PKC alpha by antisense oligodeoxynucleotide on iNOS expression via interleukin-1 receptor in vascular smooth muscle cells

Protein kinase C (PKC) family, is now classified into three groups; conventional (cPKC), novel (nPKC) and atypical (aPKC), and to date, 10 members of isozymes have been identified. We have suggested that PKC is essential to interleukin-1 (IL-1)-triggered expression of inducible NO synthase (iNOS), and that by pharmacological analysis, cPKC is not involved in iNOS induction in rat vascular smooth muscle cells (VSMC). In the present study, we identified some PKC isozymes and investigated the effect of PKC alpha knockdown by antisense oligodeoxynucleotide (AS-ODN) strategy on iNOS expression and nuclear translocation of NF-kappa B in RASMC. Western blot analysis revealed the presence of cPKC (alpha), nPKCs (delta and epsilon) and aPKCs (tau and lambda). Short-time (10-20 min) treatment with phorbol 12-myristate 13-acetate (PMA) induced translocation of PKC alpha from cytosolic to particulate fraction. PKC alpha was completely downregulated by treatment with 100 nM PMA for 24 hours. Treatment with AS-ODN against PKC alpha mRNA depleted PKC alpha specifically, and had no detectable effect on the other PKCs. The production of iNOS mRNA, but not nuclear translocation of NF-kappa B, stimulated by IL-1 beta was decreased by PKC alpha knockdown. These results suggest that there are 5 PKC isozymes in RASMC, and that PKC alpha is involved in iNOS expression triggered by IL-1 beta, supporting our previous pharmacological conclusion.     

The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death

Research over the last decade has extended the prevailing view of mitochondria to include functions well beyond the critical bioenergetics role in supplying ATP. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organelle communication, aging, many diseases, cell proliferation and cell death. Apoptotic signal transmission to the mitochondria results in the efflux of a number of potential apoptotic regulators to the cytosol that trigger caspase activation and lead to cell destruction. Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) is involved in this release of proteins via the outer mitochondrial membrane. VDAC in the outer mitochondrial membrane is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. VDAC has been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins and due to its function in the release of apoptotic proteins located in the inter-membranal space. The diameter of the VDAC pore is only about 2.6-3 nm, which is insufficient for passage of a folded protein like cytochrome c. New work suggests pore formation by homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins such as Bax or Bak. This review provides insights into the central role of VDAC in cell life and death and emphasizes its function in the regulation of mitochondria-mediated apoptosis and, thereby, its potential as a rational target for new therapeutics.     

The cardiovascular actions of clonidine and neuropeptide-Y in the ventrolateral medulla of the rat.

1. The cardiovascular responses to neuropeptide-Y (NPY) (25 and 50 pmol) and clonidine (10 and 20 nmol) were examined following microinjection into the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla (CVLM). Mean arterial pressure (MAP) and heart rate (HR) were measured in anaesthetized rats, pre- and post-injection. 2. The alpha 2-adrenoceptor agonist clonidine (10 and 20 nmol) reduced MAP and HR significantly when microinjected into the CVLM and RVLM. 3. NPY (25 and 50 pmol) microinjected into the CVLM decreased MAP and HR. However, in the RVLM neither dose had a significant cardiovascular effect. 4. The possibility of a functional interaction between the adrenergic system and NPY was examined by co-administration of clonidine and NPY in doses that gave submaximal blood pressure responses. In the CVLM this produced hypotension and bradycardia which was similar in magnitude to the sum of their individual responses, indicating that in this area their actions appear to be independent. 5. In the RVLM, where NPY has no significant cardiovascular effects, co-administration with clonidine, did not alter the response to clonidine. 6. It appears that in the areas investigated, there is no functional interaction between NPY and clonidine.     

Role of protein kinase C in BSA-AGE-mediated inducible nitric oxide synthase expression in RAW 264.7 macrophages

In the present study, the roles of protein kinase C (PKC) in BSA-derived advanced glycosylation end products (BSA-AGEs)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were investigated. Treatment of RAW 264.7 cells with BSA-AGEs caused dose- and time-dependent increases in NO release and iNOS expression in RAW 264.7 cells, whereas BSA alone had no effect on iNOS induction. The tyrosine kinase inhibitor (genistein), the phosphatidylinositol-specific phospholipase C inhibitor (U-73122), the phosphatidylcholine-specific phospholipase C inhibitor (D-609), and the PKC inhibitors (staurosporine, Ro 31-8220, and Go 6976) all inhibited BSA-AGE-induced NO release and iNOS expression in RAW 264.7 cells. Stimulation of RAW 264.7 cells with BSA-AGEs resulted in the formation of inositol monophosphate; the response was attenuated by U-73122 and genistein. BSA-AGEs stimulated PKC-alpha, -betaI, -delta, and -eta but not -zeta translocation from the cytosol to the membrane. However, incubation of RAW 264.7 cells with BSA-AGEs increased phosphorylation of PKC-zeta at threonine-410, which reflects activation of PKC-zeta, indicating the possible involvement of these PKC isoforms in AGE-mediated effects. Pretreatment of RAW 264.7 cells with U-73122, D-609, and genistein reduced the AGE-stimulated translocation of PKC-alpha, -betaI, -delta, and -eta and activation of PKC-zeta. Taken together, these data suggest that BSA-AGEs might activate PKC and subsequently induce iNOS expression and NO release.