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Qualitative Immunofluorescence Results Aliquots of cells control and stimulated ; were subjected to indirect membrane immunofluorescence in the presence of 20 mM sodium azide . Results obtained with the antiserum against glycoprotein III are shown in Fig. 1 . In unstimulated control preparations only a few cells exhibited a weak specific staining. After stimulation with carbachol a more intense staining was observed and this was even more pronounced for cells stimulated with Baz + . However, the degree of immunostaining varied considerably among individual cells Fig. 1 ; . A few cells were without staining, probably representing nonchromaffin cells . Specific immunostaining Fig. 2 ; was confined to the cell surface in tiny spots or larger patches . This was true for all cell preparations irrespective of pretreatment, including experiments where the cells were fixed with formaldehyde prior to immunofluorescence . Analogous results not shown ; were obtained with an antiserum against dopamine -hydroxylase . With preimmune serum no staining was observed . When stimulated cells were washed and incubated again for 30 min, the degree of staining decreased . Apparently the number of fluorescent patches on the cell surface became reduced . There was no evidence that the size of the remaining patches differed from those of unincubated cells. Since the decrease of fluorescence did not occur uniformly for all cells, it was difficult to evaluate this in a reliable way. Quantitative Evaluation of Immunofluorescence Results Aliquots of cells stained immunohistochemically with antiserum against glycoprotein III were evaluated by a fluorescence-activated cell sorter. Control unstimulated ; cells Fig. 3 ; show a fluorescence profile that is different from cells only treated with preimmune serum. This fluorescence profile was well reproducible, varying little from one cell preparation to. Hepatic endocrine tumors patients. The alterations blood flow were correlated responses to octreotide secretion and symptoms sequent behavior of the. 1 he phagocytosis of the shed tips of photoreceptor outer segments OS ; is one of the many important functions of the retinal pigment epithelium RPE ; . Recent evidence suggests that this is a receptor-mediated process for review see' ; , in which a receptor phagocytosis receptor ; on the surface of the RPE interacts with a ligand on the plasma membrane of the Michael O. Hall, Barry L. Burgess, OS. As with most other receptor-mediated processes, it Toshka A. Abrams, and Mayola O. Martinez is likely that binding of an OS the receptor triggers Purpose. To investigate the effect of carbachol on the an intracellular signaling event, which causes the RPE to ingest the bound OS. The target of such a signal phagocytosis of photoreceptor outer segments OS ; in cultures of normal Long-Evans and dystrophic Royal may well be the cytoskeletal elements that are abunCollege of Surgeons RCS ; rat retinal pigment epithedant in both the RPE microvilli and in the cortical lial RPE ; cells. area underlying the plasma membrane.23 Nothing is Methods. Retinal pigment epithelial cells from normal known about the signaling pathway that may operate and RCS rats were grown in tissue culture. On reaching in this phagocytic function. Previous studies from this confluence, they were presented with OS suspended in laboratory have investigated the possible role of the Krebs-Henseleit buffer in the presence or absence of protein kinase C pathway4 as well as of the protein carbachol and LiCl. The number of bound and ingested kinase A pathway5'6 as mediators of OS ingestion. HowOS was quantitated using double immunofluorescence ever, stimulation of both these pathways causes an staining. inhibition of OS ingestion, without causing a parallel Results. LiCl inhibited the ingestion of OS by more than decrease in the binding of OS. This indicates that the 90% but had no effect on the binding of OS by Longeffect of stimulating these pathways is intracellular, Evans RPE cells. The addition of carbachol further rerather than extracellular i.e. they do not affect the duced OS ingestion. Carbachol alone decreased OS innumber or the affinity of OS-binding sites on the gestion by normal RPE cells by 30% but had no effect plasma membrane ; . on OS binding. The effect of LiCl and carbachol on A well-studied model for investigating die process RCS RPE cells was similar to their effect on normal RPE of OS phagocytosis is the Royal College of Surgeons cells. Conclusions. Carbachol does not increase OS phagocyto- RCS ; rat, which suffers from an inherited retinal degeneration.7 The RPE of this animal displays limited sis in normal or RCS rat RPE cells. The phagocytic phagocytosis of shed OS tips, 8'9 with a resultant slow defect in RCS rat RPE cannot be reversed or overcome by stimulation of the IP3 pathway by carbachol. LiCl degeneration of the OS. Studies using RPE cells grown strongly inhibits the ingestion of OS by normal and by in tissue culture have demonstrated clearly that the RCS RPE cells, and this effect is enhanced by carbachol. mutation affects the ingestion phase of OS phagocytoInvest Ophthalmol Vis Sci 1996; 37: 1473-1477. sis because binding of OS occurs normally.10 This suggests that either the phagocytosis receptor is defective From the fides Stein Eye Institute, University of California at Los Angeles School of and thus is unable to transmit a signal to the cell Medicine. interior or that the intracellular signaling pathway to Supported l y National institutes of Health grants EY00046 and EY00331 and. liy a grant from the Foundation Fighting Blindness, Baltimore, Maryland MOH ; . which the receptor is linked is defective. Submitted for publication August 8, 1995; revised January II, 1996; accepted. Recent studies have suggested that the second Felmimy 26, 1996. Proprietary interest category: N. messenger involved in OS phagocytosis may be inosiHejmnl requests: Michael ; . Hall, Juks Stein Eye Institute, UClJ\ Medical. Center, tol trisphosphate IP3 ; . This is based on two lines of 100 Stein Plaza, Los Angeles, CA 90095-7008.

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In the Attleboro district, Durkin has a lot on her plate. Watching over five elementary schools, three middle schools, and a high school is a lot of responsibility. "A superintendent's job is a 24 job, " she said. Even so, Durkin feels that nine schools is the perfect size for her district. With too many schools, like the 130 in her previous Boston district, she feels she becomes a distant figure, whereas with too few there can be a lack of ideas and change. Durkin enjoys the personal contact that a district of this size allows. "I love being in the schools and working with the students, " she said. Although she never saw herself becoming "the person out in front, " Durkin did see herself in a type of helping profession, like teaching. Thanks to the encouragement of mentor Dr. Tom Payzant, however, she has become the person out in front. Payzant was the superintendent of the Boston Public Schools where Durkin was assistant superintendent. Since then, he has both encouraged her and helped her to stay on track in her superintending duties. All in all, Durkin is happy with the energy both teachers and students at AHS have, especially in doing things the right way. "I thrilled to be a part of Attleboro Public Schools, " she said. "I love working with such great staff and such great kids. Enol sulfate markedly reduced airway hyperresponsiveness to inhaled methacholine and histamine. These findings contrast with previous observations among these subjects that the response to methacholine was blocked with baclofen, oxybutynin, or ipratropium bromide, whereas the response to histamine was not affected by these agents.3, 4, 7, 8 Findings of the current study are comparable to those obtained among subjects with asthma or chronic obstructive lung disease, which have shown significant reduction in response to histamine or methacholine within minutes following inhalation of albuterol, fenoterol, or metaproterenol that persists for 4 to 6 h.13-18 Similar attenuation was observed in normal subjects with upper respiratory tract infections, 19 or those challenged with carbachol or inert dust particles.20 The protective effects of 2-agonists are attributed to interaction of the agent with 2-receptors, which are widely distributed on airway smooth muscle, thereby causing functional relaxation irrespective of the bronchoconstrictor stimulus.9, 21 Studies performed in vitro have demonstrated that activation of 2receptors on cholinergic and sensory nerves inhibits release of acetylcholine and neuropeptides, 22, 23 which suggests a potential indirect mechanism of bronchodilation since methacholine acts directly on and carbenicillin. Carbachol is somewhat stronger and echothiophate phospholine® is stronger still but has a tendency to cause cataracts and is only used in patients who have already had cataracts removed.
INFLUENCE OF CARBACB0L INJECTION INTO THE LATERAL HYPOTHALAMIC AREA LHA ; OF SPONTANEOUSLY HYPERTENSIVE RATS O N THE RENAL EXCRETION OF Na + , IT1", AND WATER. J . A C.R. S i l M.R. Furtado. Lab. of Bioraembranes, Kidney and Hypertension, U . S . School of Medicine, R i b e S.P., Brazil. The c e n nervous system e x e upon t h e excretion of water and e l e known that microinjectlons of cholinergic and adrenergic agonists into the hypothalamus intensify the natriuretic and kaliuretic responses in nonnotensive rats Silva-Netto, A.J.P. 244: F64, 1983 ; . W previously observed differing characteristics among e spontaneously hypertensive rats SHR ; and normotensive Wistar rats as far as their renal excretions of Na + and I * were T * concerned. Our sain objective was to disclose a possible interaction between hypothalamic cholinergic pathways and the development of hypertension in the SHRs. By stereotaxic means, cannulae were iaplanted into the L A of the rats 230H 270g body weight ; . After a 14-hour fasting period, a 5Z body weight water load was administered by gavage. The animals were then placed in individual metabolic cages to c o urine at 20-minute intervals, making for 7 periods. After a f i control sanple U ; , different doses of carbachol CCh ; were injected into the L A 0.125, 0.25, 0.5, and 1.0 ug vO, H while equal volumes of 0.15H NaCl were used as the s a l controls. Results were as follows: S R n-9 ; H UUt.r n-10 ; Periods urv Baul 2, 31, 1 O, 7O, 3 1, l, 30, 4 1, 0, 810, 3 2, NaCl 0.125 1, 510, 0 4, 610, 7 0 1, 910, 8 0, 410, 3 13, Values are Beans 1 S.E.M.; o - number of rats; experimental values are maximal or niniaal results according to the direction of tha observed changes; V - pl nin; DjjaV and DjV pEq min. Aa compared to the Wistar, the SHRs exhibited a smaller fractional excretion of sodiua in response to C h stimulation, suggesting a diminished s e n lesser a f f the L A to the cholinergic stimulus that H regulates renal Na * excretion. Supported by FEJEP, FAPESP and CNPq and carboplatin.

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PHINMS Binding to Dispersed Pancreatic Acini-Incubation of dispersed pancreatic acini with 250 p~ [3H]NMS ata density approximately 0.3 mg of acinar protein ml at 37 resulted in maximal binding of [3H]NMS to acini 60 min, by and bindingwas not changed significantly from 60 to 90 min Fig. lA ; . Total [3H]NMS bound to acini was about 7% of the amount of tracer added. The additionof 10 FM unlabeled NMS decreased [3H]NMS binding from 7 to 0.2% nonspecific binding ; , and this was not decreased further with greater concentrations of NMS. The incubation of acini at approximately 0.3 mg of acinar protein ml with [3H]NMS resulted in specific binding that was proportional to the tracer concentration in the 0.125-1 nM [3H]NMS range Fig. 1B ; . When tested after a 60-min incubation with 250 [3H]NMS, specific [3H]NMSbinding was increased proportional to the amountof acini incubated in the 0.1-0.5 mg of acinar protein ml range Fig. IC ; . o Competitive Binding of f'H]NMS by NMS-Competitive 0 15 30 45 inhibition curves were generated with 250 [3H]NMS and TIME rnin ; 1 to 100 nM NMS Fig. 2 ; . A Scatchard plot 18 ; of the FIG. 3. Time courses of specific binding of [3H]NMS in the data Fig. 2, inset ; was linear, indicating a single class of presence of 0.1 m carbachol with or without 10 n M CCK or M 1 TPA. Values are themean & S.E. of three separate experiments, NMS binding sites with an affinity of 0.22 k 0.04 nM and a capacity of 61.5 k 5.1 fmol mg of protein n 4 ; K, CCKand each experiment was performed in duplicate.
1 Simonneau G, Galie N, Rubin LJ, et al. Clinical classification of pulmonary hypertension. J Coll Cardiol 2004; 43 12 Suppl ; : 5S12S 2 Humbert M, Morrell NW, Archer SL, et al. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Coll Cardiol 2004; 43 12 Suppl ; : 13S24S 3 Wagenvoort CA. Lung biopsy specimens in the evaluation of pulmonary vascular disease. Chest 1980; 77: 614 Wagenvoort CA. Morphological substrate for the reversibility and irreversibility of pulmonary hypertension. Eur Heart J 1988; 9 Suppl ; : 712 5 Fuster V, Steele PM, Edwards WD, et al. Primary pulmonary hypertension: natural history and the importance of thrombosis. Circulation 1984; 70: 580 Bjornsson J, Edwards WD. Primary pulmonary hypertension: a histopathologic study of 80 cases. Mayo Clin Proc 1985; 60: 16 Pietra GG, Edwards WD, Kay JM, et al. Histopathology of primary pulmonary hypertension: a qualitative and quantitative study of pulmonary blood vessels from 58 patients in the National Heart, Lung, and Blood Institute, Primary Pulmonary Hypertension Registry. Circulation 1989; 80: 1198 Edwards BS, Weir EK, Edwards WD, et al. Coexistent pulmonary and portal hypertension: morphologic and clinical features. J Coll Cardiol 1987; 10: 12331238 Wagenvoort CA, Mulder PG. Thrombotic lesions in primary plexogenic arteriopathy: similar pathogenesis or complication? Chest 1993; 103: 844 Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest 1991; 21: 361374 Eisenberg PR, Lucore C, Kaufman L, et al. Fibrinopeptide A levels indicative of pulmonary vascular thrombosis in patients with primary pulmonary hypertension. Circulation 1990; 82: 841 Rostagno C, Prisco D, Abbate R, et al. Pulmonary hypertension associated with long-standing thrombocytosis. Chest 1991; 99: 13031305 Rich S, Hart K. Familial pulmonary hypertension in association with an abnormal hemoglobin: insights into the pathogenesis of primary pulmonary hypertension. Chest 1991; 99: 1208 Langleben D, Moroz LA, McGregor M, et al. Decreased half-life of fibrinogen in primary pulmonary hypertension. Thromb Res 1985; 40: 577580 Huber K, Beckmann R, Frank H, et al. Fibrinogen, t-PA, and PAI-1 plasma levels in patients with pulmonary hypertension. J Respir Crit Care Med 1994; 150: 929 Boyer-Neumann C, Brenot F, Wolf M, et al. Continuous infusion of prostacyclin decreases plasma levels of t-PA and PAI-1 in primary pulmonary hypertension. Thromb Haemost 1995; 73: 735736 Hoeper MM, Sosada M, Fabel H. Plasma coagulation profiles in patients with severe primary pulmonary hypertension. Eur Respir J 1998; 12: 1446 Welsh CH, Hassell KL, Badesch DB, et al. Coagulation and fibrinolytic profiles in patients with severe pulmonary hypertension. Chest 1996; 110: 710 Geggel RL, Carvalho AC, Hoyer LW, et al. von Willebrand factor abnormalities in primary pulmonary hypertension. Rev Respir Dis 1987; 135: 294 Lopes AA, Maeda NY. Circulating von Willebrand factor antigen as a predictor of short-term prognosis in pulmonary hypertension. Chest 1998; 114: 1276 Lopes AA, Maeda NY, Bydlowski SP. Abnormalities in circu chestjournal and carmustine.

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Infusion of each dose of carbachol or L-NMMA. Linear regression was performed for each plot, and r values were 0.81 to 0.99 mean 0.94 0.04 ; . Therefore, a linear model was adopted. Although volume-pressure plots varied in slope compliance ; between individuals, within individuals there was little change in slope at different stages of the study. In other words, shifts in the plots induced by infusion of the active agents were parallel and were due to changes in venous tone rather than in compliance.

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Chondrial ATP synthesis in response to GLP-1, we sought to demonstrate that mobilization of intracellular Ca# + by other mechanisms could also increase [ATP]c and [ATP]m. Supporting this view, activation of muscarinic acetylcholine receptors with carbachol [39], and thus the production of IP , led to a robust, $ but transient, increase in [Ca# + ]c and [Ca# + ]m, and sustained increases in [ATP]c and [ATP]m Figure 6 ; . In order to determine whether GLP-1-induced [Ca# + ]m increases were essential to trigger a rise in [ATP]m, we next examined the effect of chelation of intracellular Ca# + with bis o-aminophenoxy ; ethane-N, N, Nh, Nh-tetra-acetic acid tetrakis acetoxymethyl ester ; BAPTA\AM ; . Under these conditions, the ability of GLP-1 to augment [Ca# + ]c and [Ca# + ]m results not shown ; and to increase [ATP]m Figure 7A ; was completely abolished. These results suggest that the mobilization of Ca# + from an intracellular Ca# + store is essential for the subsequent elevation of [Ca# + ]m and [ATP]m by GLP-1. To explore the mechanisms involved in the GLP-1-induced activation of mitochondrial ATP synthesis in more detail, we next used the membrane-permeable IP receptor antagonist, $ xestospongin C [40, 41] and the RyR inhibitor, ryanodine. Added separately, xestospongin C 10 M ; ryanodine 10 M ; slightly reduced GLP-1-induced [ATP]m increases Figure 7A ; , whereas Chairman of carbachol holland-brown is carbachol carbachol to the business and caverject.

A paired T-test was used to compare the early and late bronchoconstrictor responses after treatment with sIL-13R or IMA-638, with the responses in the absence of drug. Comparisons were made for the peak early phase response, average early phase response time 0 4 hours ; , and average late phase response 5 8 hours ; . To assess the response to carbachol pre-Ascaris PC400 values were normalized to100% and the post-Ascaris response was determined in the presence or absence of drug. These effects were also analyzed using a paired T-test. Significance was accepted as a p value of 0.05 using a two-tailed analysis. Data in the text and figures are presented as mean s.e.m.

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In A, upper traces are excerpts from an intracellular current clamp recording from a CA3 pyramidal neurone in the presence of 1 carbachol a ; , 10 carbachol b ; and in the combined presence of 10 carbachol and 2 NBQX c ; to illustrate burst-mode activity, theta-mode activity and the slow membrane potential oscillation, respectively. The record shown directly below each trace represents the region indicated by an asterisk in the corresponding upper trace, displayed on an expanded time scale. B shows two consecutive theta-episodes recorded in another neurone at -55 and -77 mV, to illustrate that this activity is due to a bombardment of rhythmic EPSPs. In C, drop application of 10 carbachol, at the time indicated by the filled triangle and close to the recorded neurone, generates theta-mode activity without prior activation of burst-mode activity. Scale bars: 50 mV, 50 s A, upper traces 50 mV, 400 ms A, lower traces 50 mV, 25 s B 50 mV, 40 s C and cefazolin. 1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Goto K, and Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411, 1988. Koseki CM, Imai Y, Yanagisawa M, and Masaki T: Autoradiographic distribution of binding sites for endothelin in rat tissues: a neuropeptide? J Physiol 256 Regulatory Integrative Comp Physiol 25 ; : R858, 1989. 3. De Gouville A-C, Lippton HL, Cavero I, Summer WR, and Hyman AL: Minireview: Endothelin--A new family ofendothelium-derived peptides with widespread biological properties. Life Sci 45: 1499, 1989. Lovenberg W and Miller RC: Endothelin: A review of its effects and possible mechanisms of action. Neurochem Res 15: 407, 1990. Secrest RJ and Cohen ML: Endothelin effects in vascular and nonvascular smooth muscle. Life Sci 45: 1365, 1989. Van Renterghem C, Vigne P, Barhanin J, Schmid-Alliana A, Frelin C, and Lazdunski M: Molecular mechanism of action of vasoconstrictor peptide endothelin. Biochem Biophys Res Commun 157: 977, 1988. Hay DWP: Mechanism of endothelin-induced contraction in guinea-pig trachea: comparison with rat aorta. Br J Pharmacol 100: 383, 1990. MacCumber MW, Ross CA, Glasser BM, and Snyder SH: Endothelin: Visualization of mRNAs by in situ hybridization provides evidence for local action. Proc Natl Acad Sci U S A 86: 7285, 1989. Korbmacher C, Helbig H, Haller H, Erickson-Lamy KA, and Wiederholt M: Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells. Biochem Biophys Res Commun 164: 1031, 1989. Howe PH, Akhtar RA, Naderi S, and Abdel-Latif AA: Correlative Studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris. J Pharmacol Exp Ther 239: 574, 1986 and carbachol.

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Cholinergic and 1-adrenergic agonists are major stimuli of lacrimal gland secretion of protein. We have shown that these two agonists used PKC and Ca2 to various degrees to stimulate peroxidase secretion. To determine whether EGF used the same intracellular signaling pathways as cholinergic or 1adrenergic agonists, acini were incubated in the presence of each agonist alone or with combination of agonists. EGF 10 7 M ; significantly stimulated peroxidase secretion to 2.9% 0.9% from a basal value of 1.8% 0.6%. Carbachol and phenylephrine also significantly stimulated peroxidase secretion to 2.9% 0.7% of total peroxidase and 6.5% 1.1%, respectively Fig. 5 ; . Simultaneous addition of the cholinergic agonist carbachol 10 4 M ; and EGF 10 7 M ; resulted in secretion of 1.9% 0.2% Fig. 5 ; . This secretion was significantly less than the calculated theoretical secretion of 4.0% 1.0%. Simultaneous addition of the phenylephrine 10 4 M ; and EGF 10 7 M ; resulted in secretion of 4.7% 0.6%. This secretion was also significantly less than the calculated theoretical secretion of 7.4% 1.5% Fig. 5 ; . These data imply that EGF uses the and cefprozil.
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