Modulating of ocular inflammation with macrophage migration inhibitory factor is associated with notch signalling in experimental autoimmune uveitis.

The aim of this study was to examine whether macrophage migration inhibitory factor (MIF) could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and to explore the underlying mechanism. Mutant serotype 8 adeno-associated virus (AAV8) (Y733F)-chicken ß-actin (CBA)-MIF or AAV8 (Y733F)-CBA-enhanced green fluorescent protein (eGFP) vector was delivered subretinally into B10.RIII mice, respectively. Three weeks after vector delivery, EAU was induced with a subcutaneous injection of a mixture of interphotoreceptor retinoid binding protein (IRBP) peptide with CFA. The levels of proinflammatory cytokines were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Retinal function was evaluated with electroretinography (ERG). We found that the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP-injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, delta-like ligand 4 (Dll4), Notch receptor intracellular domain (NICD) and hairy enhancer of split-1 (Hes-1) increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF-injected EAU mice. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced the expression of NICD, Hes-1 and proinflammatory cytokines. Further, a MIF antagonist ISO-1 attenuated intraocular inflammation, and inhibited the differentiation of T helper type 1 (Th1) and Th17 in EAU mice. We demonstrated that over-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signalling. The expression of both MIF and its receptors are elevated in EAU mice. Over-expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signalling and Notch pathway. Our data suggest that the MIF-Notch axis may play an important role in the pathogenesis of EAU. Both the MIF signalling pathways may be promising targets for developing novel therapeutic interventions for uveitis.

Selective activation of angiotensin AT2 receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, which progressively leads to right-heart failure and death. A dys-regulated renin angiotensin system (RAS) has been implicated in the development and progression of PH. However, the role of the angiotensin AT2 receptor in PH has not been fully elucidated. We have taken advantage of a recently identified non-peptide AT2 receptor agonist, Compound 21 (C21), to investigate its effects on the well-established monocrotaline (MCT) rat model of PH. EXPERIMENTAL APPROACH: A single s.c. injection of MCT (50 mg·kg(-1) ) was used to induce PH in 8-week-old male Sprague Dawley rats. After 2 weeks of MCT administration, a subset of animals began receiving either 0.03 mg·kg(-1) C21, 3 mg·kg(-1) PD-123319 or 0.5 mg·kg(-1) A779 for an additional 2 weeks, after which right ventricular haemodynamic parameters were measured and tissues were collected for gene expression and histological analyses. KEY RESULTS: Initiation of C21 treatment significantly attenuated much of the pathophysiology associated with MCT-induced PH. Most notably, C21 reversed pulmonary fibrosis and prevented right ventricular fibrosis. These beneficial effects were associated with improvement in right heart function, decreased pulmonary vessel wall thickness, reduced pro-inflammatory cytokines and favourable modulation of the lung RAS. Conversely, co-administration of the AT2 receptor antagonist, PD-123319, or the Mas antagonist, A779, abolished the protective actions of C21. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that the AT2 receptor agonist, C21, may hold promise for patients with PH.

Efficacy of 3,5-dibromo-L-phenylalanine in rat models of stroke, seizures and sensorimotor gating deficit.

BACKGROUND AND PURPOSE: Abnormal glutamatergic activity is implicated in neurologic and neuropsychiatric disorders. Selective glutamate receptor antagonists were highly effective in animal models of stroke and seizures but failed in further clinical development because of serious side effects, including an almost complete set of symptoms of schizophrenia. Therefore, the novel polyvalent glutamatergic agent 3,5-dibromo-L-phenylalanine (3,5-DBr-L-Phe) was studied in rat models of stroke, seizures and sensorimotor gating deficit. EXPERIMENTAL APPROACH: 3,5-DBr-L-Phe was administered intraperitoneally as three boluses after intracerebral injection of endothelin-1 (ET-1) adjacent to the middle cerebral artery to cause brain injury (a model of stroke). 3,5-DBr-L-Phe was also given as a single bolus prior to pentylenetetrazole (PTZ) injection to induce seizures or prior to the administration of the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) to cause disruption of prepulse inhibition (PPI) of startle (sensorimotor gating deficit). KEY RESULTS: Brain damage caused by ET-1 was reduced by 52%, which is comparable with the effects of MK-801 in this model as reported by others. 3,5-DBr-L-Phe significantly reduced seizures induced by PTZ without the significant effects on arterial blood pressure and heart rate normally caused by NMDA antagonists. 3,5-DBr-L-Phe prevented the disruption of PPI measured 3 days after the administration of ET-1. 3,5-DBr-L-Phe also eliminated sensorimotor gating deficit caused by MK-801. CONCLUSION AND IMPLICATIONS: The pharmacological profile of 3,5-DBr-L-Phe might be beneficial not only for developing a therapy for the neurological and cognitive symptoms of stroke and seizures but also for some neuropsychiatric disorders.

Impaired glutamatergic synaptic transmission in the PKU brain.

This paper reviews recent results of our investigation of the mechanisms whereby hyperphenylalaninemia may cause brain dysfunction in classical phenylketonuria (PKU). Acute applications of L-Phe in rat and mouse hippocampal and cerebrocortical cultured neurons, at a range of concentrations found in PKU brain, significantly and reversibly depressed glutamatergic synaptic transmission by a combination of pre- and postsynaptic actions: (1) competition for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptors; (2) attenuation of neurotransmitter release; (3) competition for the glutamate-binding site of (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid and kainate (AMPA/kainate) receptors. Unlike L-Phe, its non-tyrosine metabolites, phenylacetic acid, phenylpyruvic acid, and phenyllactic acid, did not produce antiglutamatergic effects. L-Phe did not affect inhibitory gamma-aminobutyric (GABA)-ergic transmission. Consistent with this specific pattern of effects caused by L-Phe in neuronal cultures, the expression of NMDA receptor NR2A and AMPA receptor Glu1 and Glu2/3 subunits in brain of hyperphenylalaninemic PKU mice (Pah(enu2) strain) was significantly increased, whereas expression of the NMDA receptor NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Considering the important role of glutamatergic synaptic transmission in normal brain development and function, these L-Phe-induced changes in glutamatergic synaptic transmission in PKU brain may be a critical element of the neurological symptoms of PKU.

Differential modulation of glutamatergic transmission by 3,5-dibromo-L-phenylalanine.

An increasing body of evidence supports the hypothesis that diminished function of N-methyl-D-aspartate (NMDA) receptors and the associated increase in glutamate release and overstimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors are critical elements of the pathophysiology of schizophrenia. Here, we describe a halogenated derivative of the aromatic amino acid L-phenylalanine that 1) activates NMDA receptors, 2) depresses presynaptic glutamate release, and 3) blocks AMPA/kainate receptors. The experiments were conducted in rat cerebrocortical cultured neurons by using the patch-clamp technique. 3,5-Dibromo-L-phenylalanine (3,5-DBr-L-Phe) augmented NMDA miniature excitatory postsynaptic currents (mEPSCs) and activated the steady-state current, effects that were eliminated by NMDA receptor antagonists DL-2-amino-5-phosphonopentanoic acid and MK-801 (dizocilpine maleate; 5H-dibenzo[a,d]cyclohepten-5,10-imine). 3,5-DBr-L-Phe was a partial agonist at the glutamate-binding site of NMDA receptors with an EC50 of 331.6 +/- 78.6 microM and with an efficacy of 30.5 +/- 4.7% compared with NMDA. 3,5-DBr-L-Phe depressed both amplitude and frequency of AMPA/kainate mEPSCs. The IC50 of 3,5-DBr-L-Phe to inhibit AMPA/kainate mEPSC frequency was 29.4 +/- 4.3 microM. 3,5-DBr-L-Phe significantly decreased paired pulse depression of AMPA/kainate EPSCs and attenuated current activated by AMPA with higher efficacy at lower concentration of AMPA. 3,5-DBr-L-Phe neither affected GABA miniature inhibitory postsynaptic currents nor elicited action potentials. By enhancing NMDA receptor function, reducing glutamate release and blocking AMPA/kainate receptors 3,5-DBr-L-Phe represents a new type of polyvalent modulator of glutamatergic synaptic transmission with potential therapeutic applications.

Long-term changes in glutamatergic synaptic transmission in phenylketonuria.

The cellular mechanisms that underlie impaired brain function during phenylketonuria (PKU), the most common biochemical cause of mental retardation in humans, remain unclear. Acute application of L-Phe at concentrations observed in the PKU brain depresses glutamatergic synaptic transmission but does not affect GABA receptor activity in cultured neurons. If these depressant effects of L-Phe take place in the PKU brain, then chronic impairment of the glutamate system, which may contribute to impaired brain function, could be detected as changes in postsynaptic glutamate receptors. This hypothesis was tested by using a combination of liquid chromatography-mass spectrometry, patch-clamp, radioligand binding and western blot approaches in forebrain tissue from heterozygous and homozygous (PKU) Pah(enu2) mice. Brain concentrations of L-Phe were nearly six-fold greater in PKU mice (863.12 +/- 17.96 micromol/kg) than in their heterozygous counterparts (149.32 +/- 10.23 micromol/kg). This concentration is significantly higher than the K(B) of 573 microM for L-Phe to compete for N-methyl-D-aspartate (NMDA) receptors. Receptor binding experiments with [3H]MK-801 showed significant up-regulation of NMDA receptor density in PKU mice. Consistent with the depressant effects of L-Phe, expression of NMDA receptor NR2A and (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Glu1 and Glu2/3 subunits was significantly increased, whereas expression of the NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Given the role of the glutamatergic system in brain development and function, these changes may, at least in part, explain the brain disorders associated with PKU.

Desflurane and sevoflurane attenuate oxygen and glucose deprivation-induced neuronal cell death.

Neuronal cell death may occur via two pathways: those causing necrosis or those causing apoptosis. Apoptosis can be activated during periods of stress such as oxygen and glucose deprivation. Anesthetic agents such as desflurane or sevoflurane can attenuate early neuronal necrotic death, but their effect on oxygen and glucose deprivation-induced apoptosis has not been investigated. Neuronal cell cultures were prepared from neonatal rat cortex and were used between 10 and 14 days in vitro. The neuronal cell cultures were pretreated 30 minutes prior to oxygen and glucose deprivation with either desflurane or sevoflurane (N = 18). Three concentrations of each anesthetic were evaluated. The cultures were then deprived of oxygen and glucose for 30, 60, or 90 minutes. Treatment with desflurane or sevoflurane was continued during the period of oxygen and glucose deprivation. Forty-eight hours after exposure, the cells were examined for apoptosis using TUNEL and DNA gel electrophoresis. Comparisons were made to neuronal cortical cell cultures exposed to oxygen and glucose deprivation alone (N = 9). This in vitro model of oxygen and glucose deprivation was successful in producing neuronal cell death during the exposure times examined. During 30-, 60-, and 90-minute periods of oxygen and glucose deprivation, both desflurane and sevoflurane significantly ( approximately 98%) attenuated neuronal cell death regardless of concentration.

L-phenylalanine selectively depresses currents at glutamatergic excitatory synapses.

To explore the hypothesis that L-phenylalanine (L-Phe) depresses glutamatergic synaptic transmission and thus contributes to brain dysfunction in phenylketonuria (PKU), the effects of L-Phe on spontaneous and miniature excitatory postsynaptic currents (s/mEPSCs) in rat and mouse hippocampal and cerebrocortical cultured neurons were studied using the patch-clamp technique. L-Phe depressed the amplitude and frequency of both N-methyl-D-aspartate (NMDA) and non-NMDA components of glutamate receptor (GluR) s/mEPSCs. The IC(50) of L-Phe to inhibit non-NMDAR mEPSC frequency was 0.98 +/- 0.13 mM, a brain concentration seen in classical PKU. In contrast, D-Phe had a significantly smaller effect, whereas L-leucine, an amino acid that competes with L-Phe for brain transporter, had no effect on mEPSCs. Unlike GluR s/mEPSCs, GABA receptor mIPSCs were not attenuated by L-Phe. A high extracellular concentration of glycine prevented the attenuation by L-Phe of NMDAR current, activated by exogenous agonist, and of NMDAR s/mEPSC amplitude, but not of NMDAR s/mEPSC frequency. On the other hand, L-Phe significantly depressed non-NMDAR current activated by low but not high concentrations of exogenous agonists. Glycine-independent attenuation of NMDAR s/mEPSC frequency suggests decreased presynaptic glutamate release caused by L-Phe, whereas decreased amplitudes of NMDAR and non-NMDAR s/mEPSCs are consistent with competition of L-Phe for the glycine- and glutamate-binding sites of NMDARs and non-NMDARs, respectively. The finding that GluR activity is significantly depressed at conditions characteristic of classical PKU indicates a potentially important contribution of impaired GluR function to PKU-related mental retardation and provides important insights into the potential physiological consequences of impaired GluR function.

Specific inhibition of N-methyl-D-aspartate receptor function in rat hippocampal neurons by L-phenylalanine at concentrations observed during phenylketonuria.

Hippocampal N-methyl-D-aspartate receptors (NMDARs) are thought to be involved in the regulation of memory formation and learning. Investigation of NMDAR function during experimental conditions known to be associated with impaired cognition in vivo may provide new insights into the role of NMDARs in learning and memory. Specifically, the mechanism whereby high concentrations of L-phenylalanine (L-Phe) during phenylketonuria (>1.2 mM) cause mental retardation remains unknown. Therefore, the effects of L-Phe on NMDA-activated currents (I(NMDA)) were studied in cultured hippocampal neurons from newborn rats using the patch-clamp technique. L-Phe specifically and reversibly attenuated I(NMDA) in a concentration-dependent manner (IC(50) = 1.71 +/- 0.24 mM). In contrast, L-tyrosine (L-Tyr), an amino acid synthesized from L-Phe in normal subjects, did not significantly change I(NMDA). Although the L-Phe-I(NMDA) concentration-response relationship was independent of the concentration of NMDA, it was shifted rightward by increasing the concentration of glycine. Consistent with an effect of L-Phe on the NMDAR glycine-binding site, L-Phe (1 mM) did not attenuate I(NMDA) in the presence of D-alanine (10 microM). Furthermore, L-Phe significantly attenuated neither glutamate-activated current in the presence of MK-801, nor current activated by AMPA. The finding that L-Phe inhibits specifically NMDAR current in hippocampal neurons by competing for the glycine-binding site suggests a role for impaired NMDAR function in the development of mental retardation during phenylketonuria and accordingly an important role for NMDARs in memory formation and learning.

Oxygen and glucose deprivation-induced neuronal apoptosis is attenuated by halothane and isoflurane.

UNLABELLED: Both in vitro and in vivo evidence supports the reduction of early ischemic, both global and focal, brain injury by volatile anesthetics. However, the protection afforded by volatile anesthetics in later neuronal death, i.e., apoptosis, caused by global ischemia has not been investigated. We induced oxygen and glucose deprivation in neuronal cortical cell cultures prepared from newborn rats on in vitro Days 10-14. This hypoxic (PO2 <50 mm Hg) condition was maintained continuously (30, 60, and 90 min). In a separate experiment, the neuronal cell cultures were exposed to isoflurane (1.13%, 2.3%, or 3.3%) or halothane (1.7%, 3.4%, or 5.1%) before oxygen and glucose deprivation, with continued exposure to isoflurane or halothane during oxygen and glucose deprivation. After 48 h, neuronal apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling and DNA gel electrophoresis. Oxygen and glucose deprivation (30, 60, and 90 min) caused significant apoptosis of cerebral cortical cultured neurons. However, pretreatment and continued treatment during the period of oxygen and glucose deprivation with halothane or isoflurane resulted in a concentration-dependent attenuation of oxygen and glucose deprivation-induced neuronal apoptosis. IMPLICATIONS: This is the first investigation to evaluate the effect of volatile anesthetics on oxygen and glucose deprivation-induced neuronal apoptosis. Oxygen and glucose deprivation-induced neuronal apoptosis can be decreased by prior and continued administration of halothane or isoflurane.

Novel role of macrophage migration inhibitory factor in angiotensin II regulation of neuromodulation in rat brain.

Previously we determined that angiotensin II (Ang II) activates neuronal AT(1) receptors, located in the hypothalamus and the brainstem, to stimulate noradrenergic pathways. To link Ang II to the regulation of norepinephrine metabolism in neurons cultured from newborn rat hypothalamus and brainstem we have used cDNA arrays for high throughput gene expression profiling. Of several genes that were regulated, we focused on macrophage migration inhibitory factor (MIF), which has been associated with the modulation of norepinephrine metabolism. In the presence of the selective AT(2) receptor antagonist PD123,319 (10 microM), incubation of cultures with Ang II (100 nM; 1-24 h) elicited an increase in MIF gene expression. Western immunoblots further revealed that Ang II (100 nM; 1-24 h) increased neuronal MIF protein expression. This effect was inhibited by the AT(1) receptor antagonist losartan (10 microM), the PLC inhibitor U-73122 (10 or 25 microM), the PKC inhibitor chelerythrine (10 microM), and the Ca(2+) chelator 1,2-bis-[2-aminophenoxy]-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (10 microM). Taken together with our observation that MIF is expressed in the terminal fields of noradrenergic neurons (hypothalamus) and that Ang II increases the expression of MIF in this region in vivo, our data may suggest a novel role of Ang II in norepinephrine metabolism.

ANG II-mediated inhibition of neuronal delayed rectifier K+ current: role of protein kinase C-alpha.

It was previously determined that ANG II and phorbol esters inhibit Kv current in neurons cultured from newborn rat hypothalamus and brain stem in a protein kinase C (PKC)- and Ca2+-dependent manner. Here, we have further defined this signaling pathway by investigating the roles of "physiological" activators of PKC and different PKC isozymes. The cell-permeable PKC activators, diacylglycerol (DAG) analogs 1,2-dioctanoyl-sn-glycerol (1 micromol/l, n = 7) and 1-oleoyl-2-acetyl-sn-glycerol (1 micromol/l, n = 6), mimicked the effect of ANG II and inhibited Kv current. These effects were abolished by the PKC inhibitor chelerythrine (1 micromol/l, n = 5) or by chelation of internal Ca2+ (n = 8). PKC antisense (AS) oligodeoxynucleotides (2 micromol/l) against Ca2+-dependent PKC isoforms were applied to the neurons to manipulate the endogenous levels of PKC. PKC-alpha-AS (n = 4) treatment abolished the inhibitory effects of ANG II and 1-oleoyl-2-acetyl-sn-glycerol on Kv current, whereas PKC-beta-AS (n = 4) and PKC-gamma-AS (n = 4) did not. These results suggest that the angiotensin type 1 receptor-mediated effects of ANG II on neuronal Kv current involve activation of PKC-alpha.

Chronotropic effect of angiotensin II via type 2 receptors in rat brain neurons.

Previously, we determined that angiotensin II (Ang II) elicits an Ang II type 2 (AT(2)) receptor-mediated increase of neuronal delayed rectifier K(+) (I(KV)) current in neuronal cultures from newborn rat hypothalamus and brain stem. This requires generation of lipoxygenase (LO) metabolites of arachidonic acid (AA) and activation of serine/threonine phosphatase type 2A (PP-2A). Enhancement of I(KV) results in a decrease in net inward current during the action potential (AP) upstroke as well as shortening of the refractory period, which may lead to alterations in neuronal firing rate. Thus, in the present study, we used whole-cell current clamp recording methods to investigate the AT(2) receptor-mediated effects of Ang II on the firing rate of cultured neurons from the hypothalamus and brain stem. At room temperature, these neurons exhibited spontaneous APs with an amplitude of 77.72 +/- 2.7 mV (n = 20) and they fired at a frequency of 0.8 +/- 0.1 Hz (n = 11). Most cells had a prolonged early after-depolarization that followed an initial fully developed AP. Superfusion of Ang II (100 nM) plus losartan (LOS, 1 microM) to block Ang II type 1 receptors elicited a significant chronotropic effect that was reversed by the AT(2) receptor inhibitor PD 123,319 (1 microM). LOS alone had no effect on any of the parameters measured. The chronotropic effect of Ang II was reversed by the general LO inhibitor 5,8,11,14-eicosatetraynoic acid (10 microM) or by the selective PP-2A inhibitor okadaic acid (1 nM) and was mimicked by the 12-LO metabolite of AA 12-(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraynoic acid. These data indicate that Ang II elicits an AT(2) receptor-mediated increase in neuronal firing rate, an effect that involves generation of LO metabolites of AA and activation of PP-2A.

Gene expression profiling of rat brain neurons reveals angiotensin II-induced regulation of calmodulin and synapsin I: possible role in neuromodulation.

Angiotensin (Ang II) activates neuronal AT(1) receptors located in the hypothalamus and the brainstem and stimulates noradrenergic neurons that are involved in the control of blood pressure and fluid intake. In this study we used complementary DNA microarrays for high throughput gene expression profiling to reveal unique genes that are linked to the neuromodulatory actions of Ang II in neuronal cultures from newborn rat hypothalamus and brainstem. Of several genes that were regulated, we focused on calmodulin and synapsin I. Ang II (100 nM; 1-24 h) elicited respective increases and decreases in the levels of calmodulin and synapsin I messenger RNAs, effects mediated by AT(1) receptors. This was associated with similar changes in calmodulin and synapsin protein expression. The actions of Ang II on calmodulin expression involve an intracellular pathway that includes activation of phospholipase C, increased intracellular calcium, and stimulation of protein kinase C. Taken together with studies that link calmodulin and synapsin I to axonal transport and exocytotic processes, the data suggest that Ang II regulates these two proteins via a Ca(2+)-dependent pathway, and that this may contribute to longer term or slower neuromodulatory actions of this peptide.

Angiotensin II increases neuronal delayed rectifier K(+) current: role of 12-lipoxygenase metabolites of arachidonic acid.

Angiotensin II (Ang II) elicits an Ang II type 2 (AT(2)) receptor-mediated increase in voltage-dependent delayed rectifier K(+) current (I(KV)) in neurons cultured from newborn rat hypothalamus and brain stem. In previous studies, we have determined that this effect of Ang II is mediated via a Gi protein, activation of phospholipase A(2) (PLA(2)), and generation of arachidonic acid (AA). AA is rapidly metabolized within cells via lipoxygenases (LO), cyclooxygenase (COX) or p450 monooxygenase enzymes, and the metabolic products are known regulators of K(+) currents and channels. Thus in the present study, we have investigated whether the AT(2) receptor-mediated effects of Ang II on neuronal I(KV) require AA metabolism and if so, which metabolic pathways are involved. The data presented here indicate that the stimulatory actions of Ang II and AA on neuronal I(KV) are attenuated by selective blockade of 12-LO enzymes. However, the effects of Ang II are not altered by blockade of 5-LO or p450 monooxygenase enzymes. Furthermore, the actions of Ang II are mimicked by a 12-LO metabolite of AA, but 5-LO metabolites such as leukotriene B(4) and C(4) do not alter neuronal I(KV). These data indicate that the AT(2) receptor-mediated stimulation of neuronal I(KV) is partially mediated through 12-LO metabolites of AA.

Antagonism of the positive dromotropic effect of isoproterenol by adenosine: role of nitric oxide, cGMP-dependent cAMP-phosphodiesterase and protein kinase G.

We hypothesized that nitric oxide (NO) plays an important role in mediating the anti-adrenergic effect of adenosine on atrioventricular (AV) nodal conduction. In guinea-pig hearts instrumented for measurement of AV nodal conduction time (atrium-to-His bundle, A-H, interval), the NO synthase (NOS) inhibitor, l-NMMA (100 microm), reversibly inhibited 80% (P=0.009, n=6) of adenosine's anti-adrenergic action on the positive dromotropic effect of isoproterenol (0.01 microm). In parallel studies carried out in rabbit AV nodal myocytes, intracellular mechanisms whereby NO mediates the inhibitory effect of adenosine on isoproterenol-induced A-H interval shortening were studied. Adenosine (3 microm) inhibited isoproterenol-stimulated (0.1 microm) I(Ca,L)(beta -I(Ca,L)) by 46+/-6% (P<0.001, n=17). Consistent with isolated heart data, the NOS inhibitors, l -NMMA (100 microm) and L-NNA (500 microm) attenuated the effect of adenosine on beta -I(Ca,L)by 69+/-8% (P<0.001, n=16) and 69+/-7% (P<0.001, n=10), respectively. An inhibitor of NO-stimulated guanylyl cyclase LY83538 (40 microm) reduced the inhibitory effect of adenosine on beta -I(Ca,L)by 97+/-6% (P=0.004, n=15). Similarly, the non-specific inhibitor of cAMP-phosphodiesterases IBMX (50 microm) decreased the anti-adrenergic effect of adenosine by 60% (P=0.02, n=6), whereas the extracellular application of the non-hydrolyzeable cAMP analog 8-Br-cAMP (500 microm) prevented this action of adenosine. Activation of cGMP-dependent protein kinase (PKG) by CPT-cGMP (300 microm) diminished beta -I(Ca,L), but to a significantly smaller degree (16+/-4%, P=0.025, n=12) than that caused by adenosine. NO mediates the anti-adrenergic effect of adenosine on AV nodal conduction by a mechanism predominately involving activation of cGMP-dependent cAMP-phosphodiesterase and to a lesser extent activation of PKG.

Expression of angiotensin AT(1) and AT(2) receptors in adult rat cardiomyocytes after myocardial infarction. A single-cell reverse transcriptase-polymerase chain reaction study.

The effector hormone of the renin-angiotensin system, angiotensin II, plays a major role in cardiovascular regulation. In rats, both angiotensin receptor subtypes, AT(1) and AT(2), are up-regulated after myocardial infarction but previous studies failed to identify the cell types which express the AT(2) receptor in the heart. To address this question we established a single-cell reverse transcriptase-polymerase chain reaction for AT(1) and AT(2) receptors to determine whether these receptor subtypes are expressed in adult rat cardiomyocytes before and 1 day after myocardial infarction. By laser-assisted cell picking, section profiles of single cells without genomic DNA contamination were isolated. After dividing samples into two identical aliquots, polymerase chain reaction amplification for AT(1) and AT(2) receptors was carried out and polymerase chain reaction products were subjected to gel electrophoresis. Compared to control (n = 4) and sham-operated animals (n = 4), the number of cardiomyocytes expressing the AT(1) receptor mRNA 1 day after myocardial infarction (n = 4) was not changed (42% and 33% versus 45%, respectively). On the other hand, AT(2) receptor mRNA was expressed in 8% and 13%, respectively, of cardiomyocytes gained from control (n = 4) and sham-operated animals (n = 4) and in 14% isolated after myocardial infarction (n = 4). These results demonstrate for the first time that the AT(2) receptor is expressed in adult cardiomyocytes in vivo. They further suggest that the previously observed up-regulation of cardiac AT(1) and AT(2) receptors after myocardial infarction involves cell types other than cardiomyocytes.

The angiotensin II type 2 receptor: an enigma with multiple variations.

Since it was discovered ten years ago, the angiotensin II (ANG II) type 2 (AT(2)) receptor has been an enigma. This receptor binds ANG II with a high affinity but is not responsible for mediating any of the classical physiological actions of this peptide, all of which involve the ANG II type 1 (AT(1)) receptor. Furthermore, the AT(2) receptor exhibits dramatic differences in biochemical and functional properties and in patterns of expression compared with the AT(1) receptor. During the past decade, much information has been gathered about the AT(2) receptor, and the steadily increasing number of publications indicates a growing interest in this new and independent area of research. A number of studies suggest a role of AT(2) receptors in brain, renal, and cardiovascular functions and in the processes of apoptosis and tissue regeneration. Despite these advances, nothing stands out as the major singular function of these receptors. The study of AT(2) receptors has reached a crossroads, and innovative approaches must be considered so that unifying mechanisms as to the function of these unique receptors can be put forward. In this review we will discuss the advances that have been made in understanding the biology of the AT(2) receptor. Furthermore, we will consider how these discoveries, along with newer experimental approaches, may eventually lead to the elusive physiological and pathophysiological functions of these receptors.

Angiotensin II-induced decrease in expression of inducible nitric oxide synthase in rat astroglial cultures: role of protein kinase C.

Inducible nitric oxide synthase (iNOS) has been implicated as a mediator of cellular toxicity in a variety of neurodegenerative disorders. Nitric oxide, which is generated in high quantities following induction of iNOS, combines with other oxygen radicals to form highly reactive, death-inducing compounds. Given the frequency of neuronal death due to neurodegenerative diseases, cerebral trauma, and stroke, it is important to study the mechanisms of regulation of iNOS in the brain. We demonstrated previously that angiotensin II (Ang II) decreases the expression of iNOS produced by bacterial endotoxin or cytokines in cultured astroglia prepared from adult rat brain. Here, we have addressed the mechanisms by which Ang II negatively modulates iNOS. The inhibitory effects of Ang II on lipopolysaccharide-induced expression of iNOS mRNA and protein and nitrite accumulation were mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate. Down-regulation of PKC produced by long-term treatment of astroglia with phorbol 12-myristate 13-acetate abolished the inhibitory effect of Ang II on lipopolysaccharide-stimulated expression of iNOS mRNA and nitrite accumulation. Finally, the reduction of lipopolysaccharide-induced nitrite accumulation by Ang II was attenuated by the selective PKC inhibitor chelerythrine. Collectively, these data indicate a role for PKC in the inhibitory actions of Ang II on iNOS expression in cultured astroglia.

Angiotensin II decreases neuronal delayed rectifier potassium current: role of calcium/calmodulin-dependent protein kinase II.

Angiotensin II (Ang II) acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion. These actions of Ang II are mediated via Ang II type 1 (AT1) receptors and involve modulation of membrane ionic currents and neuronal activity. In previous studies we utilized neurons cultured from the hypothalamus and brain stem of newborn rats to investigate the AT1 receptor-mediated effects of Ang II on neuronal K+ currents. Our data indicate that Ang II decreases neuronal delayed rectifier (Kv) current, and that this effect is partially due to activation of protein kinase C (PKC), specifically PKCalpha. However, the data also indicated that another Ca2+-dependent mechanism was also involved in addition to PKC. Because Ca2+/calmodulin-dependent protein kinase II (CaM KII) is a known modulator of K+ currents in neurons, we investigated the role of this enzyme in the AT1 receptor-mediated reduction of neuronal Kv current by Ang II. The reduction of neuronal Kv current by Ang II was attenuated by selective inhibition of either calmodulin or CaM KII and was mimicked by intracellular application of activated (autothiophosphorylated) CaM KIIalpha. Concurrent inhibition of CaM KII and PKC completely abolished the reduction of neuronal Kv by Ang II. Consistent with these findings is the demonstration that Ang II increases CaM KII activity in neuronal cultures, as evidenced by increased levels of autophosphorylated CaM KIIalpha subunit. Last, single-cell reverse transcriptase (RT)-PCR analysis revealed the presence of AT1 receptor-, CaM KIIalpha-, and PKCalpha subunit mRNAs in neurons that responded to Ang II with a decrease in Kv current. The present data indicate that the AT1 receptor-mediated reduction of neuronal Kv current by Ang II involves a Ca2+/calmodulin/CaM KII pathway, in addition to the previously documented involvement of PKC.