Differential modes for ␤ 1 -and ␤ 2 -adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in ␤ 2 -ARs, G␣ i , protein kinase A RII␣ subunits, caveolin-3, and flotillins (caveolin functional homologues); ␤ 1 -ARs, m 2 -muscarinic cholinergic receptors, G␣ s , and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RI␣ subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface ␤ 2 -ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different ␤ 2 -AR expression levels), indicating that the fidelity of ␤ 2 -AR targeting to caveolae is maintained over a physiologic range of ␤ 2 -AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of ␤ 2 -ARs (but not ␤ 1 -ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domainspecific differences in the stoichiometry of individual elements in the ␤-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.Catecholamines act through cardiac ␤-adrenergic receptors (␤-ARs) 1 to influence the contractile state of the heart. The direct inotropic and chronotropic support provided by cardiac ␤-ARs represents a critical compensatory mechanism to preserve cardiac function during stress and/or states associated with circulatory compromise. In the hearts of most mammalian species, the physiologic effects of catecholamines are mediated by the predominant ␤ 1 -AR subtype (75-80% of the total ␤-ARs), which activates a signaling pathway involving the G sdependent stimulation of adenylyl cyclase leading to the accumulation of cAMP and protein kinase A-dependent phosphorylation of key target proteins. Cardiomyocytes also express ␤ 2 -ARs that support contractile function. Until quite recently, most studies of ␤ 2 -AR signaling in cardiomyocytes were wedded to the concept that ␤ 2 -ARs signal to the G s /cAMP pathway in a manner that is essentially equivalent to the pathway activated by ␤ 1 -ARs. However, there is evidence that ␤ 2 -ARs are not functionally redundant, including the findings that ␤ 2 -ARs ...

We sought to determine whether expression of the inducible, calcium-independent isoform of nitric oxide synthase (iNOS) contributes to the tissue damage produced by focal cerebral ischemia. The middle cerebral artery was occluded in halothane-anesthetized spontaneously hypertensive rats. Twenty-four hours later rats received intraperitoneal injections of the iNOS inhibitor aminoguanidine (100 mg/kg twice per day; n = 10) or of aminoguanidine + L-arginine (300 mg/kg four times per day; n = 7), aminoguanidine + D-arginine (n = 7), arginine alone (n = 6), or vehicle (n = 9). Drugs were administered for 3 consecutive days. Infarct volume was determined by image analysis in thionin-stained brain sections 4 days after induction of ischemia. Administration of aminoguanidine reduced infarct volume by 33 +/- 4% (P < 0.05 from vehicle; analysis of variance and Tukey's test), a reduction that was antagonized by coadministration of L- but not D-arginine. Administration of L-arginine alone did not affect infarct size (P > 0.05 vs. vehicle). In separate rats (n = 10), aminoguanidine attenuated calcium-independent NOS activity in the infarct (P < 0.05 vs. vehicle) without affecting calcium-dependent activity (P > 0.05). Aminoguanidine did not affect resting cerebral blood flow or the cerebrovascular vasodilation elicited by hypercapnia, as determined by laser-Doppler flowmetry (n = 4). We conclude that aminoguanidine selectively inhibits iNOS activity in the area of infarction and reduces the volume of the infarct produced by middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract-Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester-sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4␣-PMA, which does not activate PKC) recruited calcium-sensitive PKC␣ and novel PKC␦ and PKC⑀ to this compartment. The subcellular localization of the phorbol ester-insensitive PKC isoform was not influenced by PMA. Endothelin also induced the selective translocation of PKC␣ and PKC⑀ (but not PKC␦ or PKC) to caveolae. Multiple components of the extracellular signal-regulated protein kinase (ERK) cascade, including A-Raf, c-Raf-1, mitogen-activated protein kinase kinase, and ERK, were detected in caveolae under resting conditions. Although levels of these proteins were not altered by PMA, translocation of phorbol ester-sensitive PKC isoforms to caveolae was associated with the activation of a local ERK cascade as well as the phosphorylation of a Ϸ36-kDa substrate protein in this fraction. Finally, a minor fraction of a protein that has been designated as a receptor for activated protein kinase C resides in caveolae and (along with caveolin-3) could represent a mechanism to target PKC isoforms to cardiomyocyte caveolae. These studies identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their downstream target substrates. (Circ Res. 1999;84:980-988.)

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We studied the effect of focal cerebral ischemia on inducible (iNOS) and constitutive (cNOS) nitric oxide synthase enzymatic activities in the affected brain. The middle cerebral artery (MCA) was occluded in spontaneously hypertensive rats. Animals were killed 1, 2, 4, and 7 days later. cNOS and iNOS enzymatic activities were determined in the infarcted cortex using the assay of Bredt and Snyder. cNOS was assayed in the presence of calcium, whereas iNOS was assayed in the absence of calcium and in the presence of tetrahydrobiopterin. The validity of the iNOS assay was verified in rats treated with bacterial lipopolysaccharide. In these animals, the magnitude of the induction of iNOS enzymatic activity in lung, spleen, and brain paralleled the expression of iNOS mRNA, assessed by reverse-transcription polymerase chain reaction. After MCA occlusion, calcium-dependent (cNOS) activity was markedly reduced only in lesioned cerebral cortex at days 1-7 (p < 0.001; analysis of variance and Tukey's test). In contrast to cNOS, calcium-independent (iNOS) activity was induced substantially in the infarct (p < 0.005) but not in the contralateral intact cortex (p > 0.05). iNOS activity peaked at day 2 and was not different from baseline at day 7 (p > 0.05). No NADPH diaphorase-positive neurons were observed in the area of the lesion at days 1-7. Macrophages appeared at day 2 and invaded the infarcted tissue by day 7. At this time, numerous glial fibrillary acidic protein-positive astrocytes were observed within the lesion. The results suggest that the decline in calcium-dependent (cNOS) activity reflects loss of NOS neurons within the lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

VE-cadherin is an adhesion molecule localized at the adherens junctions of endothelial cells. It is crucial for the proper assembly of vascular structures during angiogenesis and maintaining vascular integrity. We have studied 3 monoclonal antibodies (mAbs) against murine VEcadherin that inhibit angiogenesis and tumor growth. Two of these, BV13 and 10G4, also disrupted normal vessels, resulting in severe vascular leakage, whereas the third, E4G10, did not. The goal of the current report was to identify the epitope of E4G10 and distinguish it from those of the disruptive mAbs. We mapped the epitope of E4G10 to within the first 10 amino acids of mature VEcadherin and demonstrated that conserved tryptophan residues in this sequence are required for VE-cadherinmediated trans-adhesion. The disruptive mAbs target a different epitope within amino acids 45 to 56, which structural homology modeling suggests is not involved in trans-adhesion. From our studies, we hypothesize that E4G10 can only bind the neovasculature, where VEcadherin has not yet engaged in transadhesion and its epitope is fully exposed. Thus, E4G10 can inhibit junction formation and angiogenesis but is unable to target normal vasculature because its epitope is masked. In contrast, BV13 and 10G4 bind an epitope that is accessible regardless of VE-cadherin interactions, leading to the disruption of adherens junctions. Our findings establish the immediate N-terminal region of VE-cadherin as a novel target for inhibiting angiogenesis. IntroductionCadherins are a large family of adhesion molecules involved in the formation of specific cell-cell contacts. 1 In humans, more than 80 members of the cadherin superfamily have been identified and are classified into subfamilies according to the presence of conserved domains and sequence motifs. 2 Cadherins are single-pass transmembrane glycoproteins defined by distinctive extracellular cadherin domains (ECDs) of about 110 amino acids. They mediate calciumdependent homophilic interactions and are responsible for selective cell-cell recognition and adhesion. These processes play an important role during embryonic morphogenesis and maintenance of tissue architecture. 2,3 Cadherins are subdivided according to specific sequence features; subfamilies include the type I (eg, N-, E-, and C-) and type II (eg, VE-and MN-) cadherins. Both type I and type II cadherins consist of 5 ECDs (ECD1-5) and are anchored to the actin cytoskeleton through their cytoplasmic tail. 4 The determinants of adhesion and adhesive specificity among the type I classic cadherins, specifically the N-, E-and C-cadherins, have been extensively studied. [5][6][7][8][9][10] In particular, the 3-dimensional structures of the N-terminal domains of N-and E-cadherins as well as the entire 5 ECDs of C-cadherin have been solved. These data indicate that the crucial adhesive determinants reside in the N-terminal ECD1 with the central feature being a conserved tryptophan residue (W2) that inserts into the hydrophobic core of the partner cadherin molecule pres...

We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [3H]L-arginine into [3H]L-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than approximately 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.

Previous work has shown that puerarin (Pur), extracted from the dried root of Pueraria lobata (Wild) Ohwi, increases cerebral blood flow in dogs and attenuates cerebral and spinal cord injury resulting from ischemia and reperfusion in rats and rabbits. The present study further demonstrates the neuroprotective effects of Pur on cerebral ischemic injury in rats and the mechanisms underlying the protective effects. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAo) for 50 min followed by reperfusion for 24 h. Pur (50, 100 mg/kg, i.p) was administered at the onset of MCAo. Twenty-four hours after reperfusion, neurological deficits were evaluated in Pur- and vehicle-treated rats. The infarct volume and edema ratios were assessed from stained brain slices. The results showed that Pur (100 mg/kg) markedly decreased the infarct volume by 34 % ( P < 0.01) in cerebral cortex and improved the neurological functions ( P < 0.05) after MCAo. Furthermore, flow cytometric analysis of annexin-V and PI labeling cells showed that the percentages of apoptosis and necrosis in the dorsolateral cortex were significantly reduced by 38.6 % and 28.5 % ( P < 0.01 and P < 0.05) following treatment with Pur (100 mg/kg) in MCAo rats. Caspase-3 activity, a biochemical marker of apoptosis, was significantly inhibited after treatment with Pur in the dorsolateral cortex. In agreement with this result, the expression of the X-chromosome-linked inhibitor of apoptosis protein (XIAP) was obviously up-regulated after administration of Pur (100 mg/kg), while caspase-3 gene was down-regulated in the dorsolateral cortex. These results suggest that the neuroprotection of puerarin against cerebral ischemia is associated with anti-apoptosis.