Cerebral revascularization by omental free flap using contralateral superficial temporal vessels as recipient vessels: case report

BACKGROUND: Although the most common technique of cerebral revascularization is superficial temporal artery to middle cerebral artery bypass, we occasionally encounter a situation in which the ipsilateral superficial temporal artery is not available. Treatment may require several techniques including long vein graft bypass. METHODS: A 54-year-old man experienced transient ischemic attacks, and cerebral angiography revealed occlusion of the right common carotid artery. Cerebral blood flow study revealed reduced perfusion reserve capacity of the right cerebral hemisphere. We applied an omental free flap to the brain surface using the contralateral superficial temporal vessels as recipient vessels. RESULTS: Cerebral blood flow study revealed improvement of perfusion reserve capacity. Cerebral angiography revealed good collateral circulation from the omentum to the brain. The patient has not experienced a transient ischemic attack, following additional ligation of the occipital artery 13 months after the first operation. CONCLUSIONS: Because an omental flap has a long pedicle and its circulation can be monitored easily, this method is safe and as effective as a long bypass graft in a patient such as ours in whom the ipsilateral superficial temporal artery is not available for anastomoses.     

Primary cell cultures from murine kidney and heart differ in endosomal pH

Peptides from the intracellular regions of G protein-coupled receptors are useful probes of receptor-G protein coupling mechanisms. As a first step toward the genetic delivery of such "G protein inhibitors," we describe inhibition of angiotensin II (AII) receptor responses by expressed fragments of the second and third intracellular loops of the AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human embryonic kidney 293 cells with DNA encoding the rat AT1a receptor resulted in AII-dependent increases of inositol phosphates (maximum 4.5-fold). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 for AII stimulation of phospholipase C activity 5-fold (from 0.18 nM to 0.99 nM, n = 12, P < .001) and 3-fold (from 0.38 nM to 1.2 nM, n = 8, P < .002), respectively. The combined effect of AT1a/i2 and AT1a/3 was additive, and transfection of an alpha-1b adrenergic receptor third intracellular loop (alpha1b/i3) fragments also increased the EC50 for AII. Neither AT1a/i1 nor C-terminus (AT1a/Ct) constructs had significant effects on angiotensin responses. These data confirm a role for the second and third intracellular loops in angiotensin receptor responses and show the potential of this approach to blocking multiple phospholipase C-linked receptors.     

Selective reconstitution of gastrin-releasing peptide receptor with G alpha q

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor-ligand interaction. To explore these mechanisms in greater detail, we have developed an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTP gamma S to purified G protein alpha subunits. Binding studies with 125I-labeled [D-Tyr6]bombesin(6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 1.4 nM +/- 0.4 (mean +/- SD, n = 3) and capacity of 15-22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin > or = GRP > > neuromedin B. Reconstitution of urea extracted membranes with a purified G alpha q showed that receptor-catalyzed binding of GTP gamma S was dependent on agonist (GRP) and G beta gamma subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V., et al. (1994) Mol. Pharmacol. 46, 235-245]. The apparent Kd for bovine brain G beta gamma in this assay was 60 nM, and the Km for squid retinal G alpha q was 90 nM. The GRPr-catalyzed binding of GTP gamma S is selective for G alpha q, since we did not detect receptor-catalyzed exchange using either G alpha i/o or G alpha t. These data demonstrate that GRPr can functionally couple to G alpha q but not to the pertussis toxin-sensitive G alpha i/o or retinal specific G alpha t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.     

2',3'-O-(2,4,6- trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP)--a nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels

1. P2X receptor activation by alpha,beta-meATP evoked inward currents in acutely dissociated rat mesenteric artery smooth muscle cells and contractions of whole artery rings. 2. The selective P2X1 and P2X3 receptor antagonist TNP-ATP inhibited P2X receptor mediated inward currents in response to 3 microM alpha,beta-meATP (an approximately EC90 concentration) with an IC50 of approximately 2 nM. This provides further evidence that the P2X receptor underlying membrane depolarisation associated with P2X receptor activation can be accounted for by the expression of P2X1 receptors. 3. TNP-ATP inhibited alpha,beta-meATP induced contractions with an IC50 of approximately 30 microM and had non-specific effects on smooth muscle contraction. 4. The reduced potency of TNP-ATP in whole tissue experiments probably reflects the breakdown of TNP-ATP by nucleotidases. Thus, TNP-ATP is of limited use in whole tissue experiments as a P2X receptor antagonist.     

Thrombin receptor-mediated increase of two matrix metalloproteinases, MMP-1 and MMP-3, in human endothelial cells

Matrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix components and are secreted by a variety of cells including human endothelial cells. Because alpha-thrombin is known to interact with matrix components and has been shown to activate latent MMP-2 in human umbilical vein endothelial cells, we investigated whether human alpha-thrombin could also regulate other MMPs secreted by the human saphenous vein or mammary artery endothelial cells (EC). After treatment of EC with increasing concentrations of thrombin for different periods of time, a significantly higher gelatinolytic activity of both MMP-1 and MMP-3 was observed in addition to MMP-2 activation. The effect of thrombin was time and dose-dependent, reaching a maximum at 24 hours. After treatment with 5 NIH U/ml thrombin for 24 hours, Western blotting revealed 9.5- and 4.4-fold increases over control values for MMP-3 and MMP-1, respectively. The synthetic thrombin receptor agonist peptide SFLLRNPNDKYEPF fully reproduced the action of thrombin, whereas chemical inactivation of the catalytic site of thrombin abolished its effect on MMP-1 and MMP-3. Thrombin and SFLLRNPNDKYEPF both induced MMP-3 mRNA synthesis but had no significant influence on constitutive MMP-1 mRNA levels. These results demonstrate that thrombin not only activates latent MMP-2 but also modulates MMP-1 and MMP-3 production in EC, this latter effect being mediated by the G-protein-coupled thrombin receptor. Hence, our present data provide evidence to support the suspected role of thrombin in tissue remodeling and angiogenesis.     

Effects of thrombin and thrombin receptor activating peptides on rat aortic vascular smooth muscle

The role of thrombin receptor activation in isolated rat aortic rings was examined. The human thrombin receptor activating peptides (TRAPs) SFLLRNPNDKYEPF (TRAP1-14), SFLLRNP (TRAP1-7) and rat TRAP1-7 (SFFLRNP) all caused concentration-related (0.1-100 microM) contractions of endothelium-rubbed rat aortic rings. Reversal of the first two amino acids in TRAP1-14 ("reverse TRAP1-14") resulted in total loss of activity. The contractions caused by the TRAPs were reduced substantially in endothelium-intact rings due to endothelium-derived relaxing factor release because the reduced contractions were reversed by N omega-nitro-L-arginine or methylene blue. Contractions were significantly but only slightly enhanced by alpha receptor blockade and were not affected by thromboxane- or endothelin-receptor blockade or by cyclooxygenase inhibition. TRAP1-7 had no effect on contractile responses to norepinephrine, serotonin, angiotensin II or endothelin-1; however, pretreatment with nifedipine or removal of extracellular Ca++ markedly inhibited the contraction. Neither human nor rat alpha-thrombin had any contractile effect on rat aortic rings. In cultured rat aortic smooth muscle cells, alpha-thrombin (EC50 = 1.9 +/- 0.7 nM), TRAP1-14 (EC50 = 30 +/- 4 microM) and TRAP1-7 (EC50 = 20 +/- 9 microM) caused concentration-dependent increases in intracellular calcium [Ca++]i, whereas reverse TRAP1-14 was ineffective. The effect of thrombin on [Ca++]i was abolished by the thrombin inhibitor MD-805, whereas the responses to TRAP were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective interactions of mu-opioid receptors with pertussis toxin-sensitive G proteins: involvement of the third intracellular loop and the c-terminal tail in coupling

A cDNA encoding the rat mu-opioid receptor was expressed stably in a Rat-1 fibroblast cell line. Expression of this receptor was demonstrated with specific binding of the mu-opioid selective ligand [3H][D-Ala2,N-MePhe4,Gly5-ol]-enkephalin ([3H]DAMGO). In membranes of clone mu11 cells DAMGO produced a robust, concentration-dependent stimulation of basal high affinity GTPase activity. Cholera toxin-catalyzed [32P]ADP-ribosylation in membranes of this clone labelled a 40 kDa Gi family polypeptide(s) that was markedly enhanced by the addition of DAMGO. Antisera against Gi2alpha and Gi3alpha were both able to immunoprecipitate a [32P]-radiolabelled 40 kDa polypeptide(s) from DAMGO and cholera-toxin treated membranes of clone mu11, indicating that the mu-opioid receptor was able to interact effectively with both Gi2 and Gi3 in Rat-1 fibroblasts. A series of peptides derived from the delta-opioid receptor sequence were assessed for their ability to modify agonist-stimulated G protein activation and [3H] agonist binding to the receptor. In membranes from the clone mu11, specific binding of [3H]DAMGO was reduced by peptides corresponding to the NH2-terminal region of the third intracellular loop (i3.1) and the carboxyl-terminal tail (i4) of this receptor. Agonist stimulated GTPase activity and DAMGO dependent cholera toxin-catalyzed [32P]ADP-ribosylation were inhibited by peptides derived from the proximal (i3.1) and the distal portion (i3.3) of the third intracellular loop. Peptide i3.1 also inhibited DAMGO-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTP-gammaS) binding in the same membranes. In contrast, peptides derived from the second intracellular loop were without any effect.     

Effects of chronic lithium and carbamazepine treatment on G-protein subunit expression in rat cerebral cortex

Although lithium and carbamazepine (CBZ) are effective in the treatment of bipolar affective disorder, their mechanism of action is still unknown. Recent evidence suggests that lithium and CBZ might exert their therapeutic effects by modulating the function of guanosine triphosphate (GTP)-regulatory (G) proteins associated with central nervous system second messenger systems. In the present study, we showed that chronic lithium administration decreases G alpha s, G alpha i1, and G alpha i2 messenger RNA (mRNA) abundance by 25%-30% in rat cerebral cortex. However, the levels of G alpha s, G alpha i1, and G alpha i2 mRNA were unaffected by chronic CBZ treatment. The effects of lithium on G alpha s, G alpha i1, and G alpha i2 mRNA levels appear to be selective, as the mRNA levels of G alpha o, G alpha x, G beta 1, G beta 2, and G beta 3 subunits remained unchanged. Two days after terminating chronic lithium treatment, changes in G alpha s, G alpha i1, and G alpha i2 mRNA levels were not demonstrable. Short-term administration of lithium (2 days), however, reduced only the G alpha i2 mRNA levels. Surprisingly, there was no significant difference in the amount of immunologically detectable G alpha s-s, G alpha s-1, G alpha i(1 + 2), G alpha 0, and G beta (1 + 2) in the cortex of rats chronically treated with lithium or CBZ, compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anxiogenic-like consequences in animal models of complex partial seizures

Extracellular adenosine triphosphate (ATP) plays an important role in the regulation of endothelial function. However, its receptors and their signal-transduction pathways in major cerebral arterial endothelial cells are largely unknown. This study was undertaken functionally to classify the P2 purinoceptors in cultured bovine middle cerebral artery endothelial cells by using [Ca2+]i microfluorimetry. The rank order of potency to increase [Ca2+]i was 2-methylthio-ATP approximately ATP approximately uridine triphosphate (UTP) > adenosine diphosphate (ADP) > adenosine monophosphate (AMP) > alpha,beta-methylene-ATP > adenosine, suggesting that the effect was mediated by both P2y and P2u receptors. ATP, 2-methylthio-ATP, and UTP mobilized Ca2+ from intracellular stores and triggered Ca2+ entry. The effects of ATP, 2-methylthio-ATP, and UTP were reduced by phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), but only the effects of ATP and UTP were attenuated by pertussis toxin, indicating that P2y and P2u receptors may activate the same effector mechanisms by coupling to different G proteins. The [Ca2+]i entry caused by UTP was significantly reduced by the receptor-regulated Ca2+ channel blocker SK&F 96365, by P-450 inhibitor econazole and by inorganic Ca2+ entry blocker lanthanum. P2-receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive blue 2 reduced the effects of ATP and 2-methylthio-ATP, but not those of UTP, in a concentration-dependent manner. These studies suggest a coexistence of P2y and P2u receptors in cultured bovine middle cerebral artery endothelial cells.     

Progressive intracranial aneurysmal disease in a child with progressive hemifacial atrophy (Parry-Romberg disease): case report

Intracranial aneurysms are uncommon in children, and their presence often leads to suspicion of a systemic connective tissue disorder. We describe the case of a young male patient with progressive hemifacial atrophy (Parry-Romberg disease) and multiple intracranial aneurysms, a previously undescribed association, and propose that a neural crest defect may be the underlying abnormality in this patient. At age 5 years, the patient was treated for a giant aneurysm of the left cavernous carotid artery with carotid ligation in the neck and a superficial temporal artery-middle cerebral artery bypass. At age 12 years, the patient was similarly treated for a giant aneurysm of the right cavernous carotid artery, which had progressed from a previously noted minute dilatation at age 5 years, with carotid ligation and a superficial temporal artery-middle cerebral artery bypass. At age 21 years, the patient was endovascularly treated for a de novo saccular aneurysm of the left posterior cerebral artery at the P1-P2 junction and a fusiform aneurysm of the distal left posterior cerebral artery. Various studies have suggested that the facial dermis, the subcutaneous tissues, and the skeleton, as well as the tunica media of the cervicocephalic arteries, all arise from neural crest cells, and a disorder of neural crest migration might explain the constellation of findings in this patient.     

Brain alpha 2-adrenergic receptor binding during incomplete cerebral ischemia in the rat

alpha 2-Adrenergic agonists decrease sympathetic activity and improve outcome from brain ischemia. We evaluated whether changes in alpha 2-adrenergic receptor binding activity may be important in the sympathetic depressant and cerebral protective effects of halothane (1.1% inspired) or isoflurane (1.4% inspired) compared to fentanyl/nitrous oxide (N2O) anesthesia. Brain alpha 2-adrenergic receptor binding was measured using [3H]-clonidine in each of four treatment conditions: 1, unanesthetized; 2, anesthetized (fentanyl/N2O, halothane, or isoflurane): 3, anesthetized with ischemia; 4, after 4 h recovery from ischemia. Ischemia was produced by right carotid artery ligation combined with hemorrhagic hypotension to 30 mm Hg for 30 min. Both halothane and isoflurane decreased alpha 2-adrenergic receptor density 20% compared to unanesthetized values (P < 0.01). This decrease was attenuated in ischemic tissue. There were no consistent changes in receptor affinity. These results suggest that inhaled anesthetics decrease the number of alpha 2-adrenergic receptors. This decrease appears to be unrelated to plasma catecholamine concentrations but may be influenced by the degree of ischemia.     

Postnatal development of multiple opioid receptors in the spinal cord and development of spinal morphine analgesia

The postnatal ontogeny of mu, delta and kappa opioid receptor binding sites in the spinal cord of rat pups at various postnatal days was determined using in vitro autoradiographical methods. The functional effect of spinal morphine was also assessed using in vivo electrophysiological methods in rats at P14, P21 and adults (P56). Both mu and kappa opioid receptor binding-sites are present from P0 and spread relatively diffusely throughout the spinal cord. Overall binding peaks at P7 and subsequently decreases to adult levels with the mu opioid receptor binding sites regressing to become denser in the superficial dorsal horn. delta Opioid receptor binding was first seen at P7, and no distinction between superficial and deeper laminae was seen. In the adult, the relative proportions of the opiate receptors in the superficial dorsal horn are 63%, 22% and 15%, for mu, delta and kappa receptor binding sites, respectively. C-fibre evoked dorsal horn neuronal responses recorded from anaesthetized rat pups were highly sensitive to spinal morphine at P21, (EC50 0.005 microgram), compared to the adult (EC50 0.9 microgram). However, the EC50 (0.2 microgram) at P14 was greater than at P21 despite the fact that mu receptor binding was greater at P14. Opioid receptor binding is developmentally regulated and undergoes substantial postnatal reorganization. However, the number of mu receptor binding sites appears not to be the only determinant of functional sensitivity to spinal morphine. Other factors, such as coupling of the receptors are likely to be important.     

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) receptor isoform coupled to adenylate cyclase stimulation

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) (h5-HT7(a)) receptor isoform was performed using stably transfected LM(tk-) cells. Expression levels of the h5-HT7(a) receptor determined from saturation studies using either a labeled agonist ([3H]5-HT) or antagonist ([3H]LSD) were very similar (Bmax = 160-190 fmol/mg protein), suggesting that all receptors may exist in the high affinity (G protein-coupled) state. In intact cells, 5-HT produced a concentration-dependent elevation of intracellular cAMP levels ([cAMP]i) with an EC50 value of 80 nM and a maximal response of 5-fold increase above basal levels. The rank order of agonist potencies in the second messenger assay paralleled their rank order of binding affinities: 5-carboxamidotryptamine > 5-hydroxytryptamine >/= 5-methoxytryptamine > 8-hydroxy N,N-dipropyl aminotetralin > sumatriptan. Agonist potencies (EC50 values) to stimulate [cAMP]i were more than 25-fold lower relative to their respective binding affinities (Ki values) obtained in [3H]5-HT competition assays. In contrast, antagonist potencies (Kb values) to block 5-HT-stimulated [cAMP]i were in close agreement with their corresponding Ki values. These data may indicate low efficiency of receptor-effector coupling to adenylate cyclase stimulation. Pretreatment of stably transfected cells with cholera toxin abolished the 5-HT-mediated elevation of [cAMP]i, indicating that the 5-HT7(a) subtype directly interacts with Galphas protein(s) to activate adenylate cyclase(s). Clonal cell lines stably expressing h5-HT7 receptor isoforms will serve as valuable cellular models to study their function and regulation, as well as assist in the development of selective 5-HT7 receptor agents to uncover the biological roles and potential therapeutic applications of this novel receptor subtype.     

Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.     

Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.     

Transdural anastomosis in a juvenile form of moyamoya disease

INTRODUCTION: Moya-Moya disease has a low prevalence and world-wide distribution, with greater incidence in the first 5 years of life (juvenile form) or in the third decade (adult form). Stenosis of the intracranial portion of the internal carotid artery leads to secondary establishment of intracranial compensating anastamoses at different levels (leptomeninges, basal ganglia and transdural). We present the case of a 7 year old boy who presented clinically with choreiform movements and later had a cerebral infarct. On angio-MR and MR studies we observed the typical smoky image together with development of additional compensatory transdural anastomoses from the superficial temporal and ophthalmic arteries to the anterior and middle cerebral arteries.     

Evaluating the effect of superficial temporal artery to middle cerebral artery bypass on pure motor function using motor activation single photon emission computed tomography

OBJECTIVE: We evaluated and analyzed the effect of superficial temporal artery to middle cerebral artery bypass for internal carotid artery occlusion on pure motor function using motor activation single photon emission computed tomography. METHODS: Motor activation single photon emission computed tomographic (SPECT) images were obtained for nine patients who had undergone superficial temporal artery to middle cerebral artery anastomosis for symptomatic internal carotid artery occlusion. All motor activation SPECT images using the finger opposition task on the affected side were obtained before bypass surgery and at 1 week, 1 month, and 3 months after bypass surgery. The results of motor activation single photon emission computed tomography were expressed as negative or positive. RESULTS: Before bypass surgery, the resting SPECT images revealed reduction of cerebral blood flow (CBF) on the affected side in all nine patients. The results of motor activation single photon emission computed tomography in three patients were positive. One week after bypass surgery, the results of the resting and motor activation CBF studies did not demonstrate any marked changes. One month after bypass surgery, the resting CBF increased in four patients. The results obtained for two of the patients revealed preoperative positive motor activation. The results of motor activation single photon emission computed tomography obtained for five patients were positive. Three months after bypass surgery, eight patients experienced improvement in the resting CBF, and the results of motor activation single photon emission computed tomography obtained for seven patients were positive. Among these, the results of preoperative motor activation single photon emission tomography obtained for four patients were negative. CONCLUSION: Superficial temporal artery to middle cerebral artery bypass is useful not only for resting CBF but also for pure motor function based on motor activation SPECT images. From the preoperative motor activation study, it was concluded that patients with preoperative positive motor activation could attain the effect of bypass earlier than patients with preoperative negative motor activation.     

Does additional brachytherapy improve the effect of external irradiation? A prospective, randomized study in central lung tumors

A 28-residue peptide (peptide G), derived from the C-terminal (W643-S670) of the beta-adrenergic receptor kinase (betaARK), was previously identified as the critical domain for binding to the betagamma subunits of the heterotrimeric guanine-nucleotide-binding regulatory protein (G betagamma). We observed that the 18-amino-acid core of this domain is poorly conserved between betaARK1 and betaARK2 and so may provide the basis for differences in G betagamma-binding properties. Specific antibodies raised against 18-residue peptides derived from the divergent sequences (peptides P1 and P2 for betaARK1 and betaARK2, respectively) competitively inhibited G betagamma-activation of the related betaARK subtype, confirming the involvement of this region in binding to G betagamma. Peptides P1 and P2 inhibited G betagamma-stimulated activity of both betaARK1 and betaARK2, with P2 being significantly more potent than P1 (IC50 of 179+/-5 microM for P2 and >500 microM for P1). The 28-residue peptides G showed the same relative inhibitory activities (IC50 = 48+/-5 microM for G2 and 146+/-8 microM for G1). This relative order of potency G2 > G1 approximately P2 > P1 was confirmed in a direct G betagamma-binding assay. No binding selectivity for the beta1, beta2, beta3 and beta4 G beta subtypes was observed. The EC50 value for G betagamma-activation of betaARK1 was about double of that for betaARK2, indicating a higher affinity between G betagamma and betaARK2, which is the expected result based on the findings with the peptides. These findings show that the 18-residue peptides P represent the shortest sequence of betaARK that can bind to G betagamma and provide a demonstration of a functional difference between the G betagamma binding domains of betaARK1 and betaARK2.     

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells

1. The effects of extracellular adenosine 5'-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5'-triphosphate (2MeSATP) and uridine 5'-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.     

G-protein expression in melanotropes changes coincident with innervation of the developing rat pituitary intermediate lobe

The two isoforms of the dopamine D2 receptor, the D2short and the D2long differ in a 29 amino acid insert in the third cytoplasmic loop with which G proteins interact. We have previously reported that in rat melanotropes, expression of D2short increases markedly at the end of the first postnatal week which is concurrent with innervation of the intermediate lobe. Using immunohistochemistry, this study examined expression of G alpha i1/2, G alpha i3, G alpha o and G alpha s proteins before and after dopaminergic innervation. G alpha i3 increased through gestational day 20, and then remained level to postnatal day 6. At this time, coinciding with the induction of D2short expression, G alpha i3 immunoreactive intensity increased markedly, possibly indicating co-regulation of these proteins. On postnatal day 6, G alpha s immunoreactive intensity increased in some, but not all, melanotropes. The resulting heterogeneity in Gs expression persisted in the adult. G alpha i1/2 immunoreactivity did not change and G alpha o was detected only subsequent to the event of innervation. Thus, dopamine released from axons and acting through D2 receptor stimulation could increase G alpha i3 immunoreactivity and decrease G alpha s immunoreactive intensity in some melanotropes.