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Nitric oxide-mediated vascular hyper-responsiveness to endothelin
AIO00
AGA A B S T R A C T S
PHOSPHOLIPASE Ao,ALONE A~kUJASSOCEATED NITH CALCI~;I, EXPLOSIVELY ACTTVATES CARBONIC AM~YDRASE If(CA II), WHILE INDOb~THACIN(IMC) ANTAGONIZES THIS EFFECT. l°Pu~ca~,Maroela Colt~u,Gabriela Domu~a,Center for Medical Research and Assistance,Simleu Silvaniei, ROMANIA. Considering the importance of phospholipase A~, a cytosolic enzyme wlth a central role in the formation of arachidonic acid,and the role of CK II in the modulation of intracellular pH,we s~udied the effect of phospholipase A~on CA.We also followed the effect on CA I and CA~II induced by associ ation of calcium and IMC to phospholipase A2o METHOD:we followed in vitro the effect on purified ~ n d CA II induced by phospholipase Agand by e quimolar association of:phospholipase A~+Ua;phospholipase Ag+INC;phospholipase A2+Ca+IMC,Assessmen ts were perTormed according to dose-re~oonse relationship,at concentrations between lO- LIO-5M°CA activity was determined by a stopped-flow method (lihalifah RG~1971). RESULTS: Donc° Phospho" Phosphol~- Phosp'ho- Phospholi(M) lipase A 2 pase A2+Ca lipase A 2 pase A2+ +IMC +Ca+IMC IO-~ 26% 4'5% 12% 14% lO-~ 83% 137% 20% 23% lO -6 117% 198% 29% 31% 10-5 180% 300% 33% 35% DISOUSSlON:phospholipase A 2 strongly activates CA II;association of Ca induces an explosive activation of CA II,while CA I activity remains unmodifi ed. The results suggest that the lowering of intracellular pH induced by cytosolic CA II activation could favour the synthesis by phospholipase A.o of arachidonic acid,precursor of prostaglandins.~ntagonization of these activating effects by IMC suggests their acting upon on the common situs of CA II. Thus IMC seems to inhibit not only cyclooxygena se and prostaglandin synthesis,but also phospholipase A 2 and the synthesis of arachidonic acid,
• SUPPRESSION OF VASOACT1VE INTESTINAL PEPTIDE (VIP)INDUCED INCREASES IN ILEAL ION TRANSPORT BY SIGMA LIGANDS. R.K. Rao, P.J.M. Riviere, X. Pascaud, J.L. Junien and F. Porreca. Department of Pharmacology, University of Arizona, Tucson, Arizona, U.S.A and Institut de Recherche Jouveinal, France. Our recent studies have shown that JO 1784 (a o receptor agonist) and neuropeptide Y (NPY) produce net proabsorptive/antisecretory effects in mouse ileum. As most of diarrheas are mediated by hypersecretion of prosecretory agents such as prostaglandinsand VIP, we tested the effects of JO 1784 and NPY on VIP-induced changes in mouse ileal ion transport. Methods: Ion-transport was studied by measuring short circuit current (I,~) and potential difference (PD) across mouse ileum mounted in Ussing chambers in vitro. I,o and PD were recorded before and after serosal application of cumulative doses of VIP. JO 1784 or NPY mediated inhibition and reversal of VIP-induced increase of I,~ were evaluated. Involvement of VIP receptors was determined by evaluating the effects ofVIP-antagonists (VIPA-1 and VIPA-2)] on VIP, JO 1784 and NPY activities. Results: Serosal application of VIP (1-100 riM) produced sustained increase in basal Isc without altering tissue conductance; this activity was concentration-related, with As0 (95% C.L.) of 0.8 (0.3-1:1) nM. Pretreatment of tissues with JO 1784 (10 gM) or NPY (10 nM) (which produced sustained decrease in basal I~) blocked the VIP-induced increase in Isc, resulting in a respective rightward displacement in VIP concentrationresponse curve of 44 or 13 folds. JO 1784 and NPY were also reversed the effect of VIP (10 nM) in a concentration-related manner. Serosal VIPA-1 or VIPA-2 (both 3 gM) produced a slight decrease (15-19%) in basal I,o and significantly reduced the effects of VIP (10 nM) and JO 1784 (10 p.M) without altering the actions of NPY. Conclusion: The findings that VIP antagonists can inhibit the action of JO 1784 suggest that o receptor mediated regulation of ion transport in mouse ileum may involve suppression of tonically released VIP. Thus, o-agonists may prove useful in controlling diarrheas caused by VIP hypersecretion. The reduced potency of NPY in blocking VIP activity, and its insensitivity to V1P antagonists suggest that NPY and JO 1784 may involve different types of receptors.
GASTROENTEROLOGY, Voh 1 0 8 , NO. 4
• EPIDERMAL GROWTH FACTOR (EGF) REVERSES OPIOID DELTA, BUT NOT OPIOID MU OR KAPPA, RECEPTOR-MEDIATED CHANGES IN ILEAL ION TRANSPORT BY A TYROSINE KINASE (TK)-DEPENDENT MECHANISM. ILK. Rao and F. Porreca. Department of Pharmacology, University of Arizona, Tucson, AZ 85724. Our previous studies have shown that EGF produced an increase in shortcircuit current (I~) in mouse ileum in which ganglionic activity was suppressed by ganglionic blockade. As opioids act at a pre-ganglionic site to regulate intestinal ion transport, the activity of EGF was tested in tissues pretreated with opioids with selectivity for 6 ([D-Ala2,Glu4]deltorphin), p. (DAMGO) and K (U50,488H) receptors. EGF activity was also tested in tissues treated with other antisecretory/proabsorptive agents, such as neuropeptide Y (NPY) somatostatin (S-14) or norepinephrine (NE). Methods: Ion-transport was studied by measuring the I,~ and potential difference (PD) across mouse ileal sheets mounted in Ussing chambers in vitro. Effects oflumir/ally or serosally applied mouse EGF on Is~ and PD were evaluated in tissues pretreated with serosal [D-Ala2,Glu4]deltorphin (100 nM), DAMGO (1 gM), U50,488H (3 p.M), NPY (30 nM), S-14 (1 gM) or NE (1 gM); all these compounds produced sustained decrease in ileal I~. Results: Serosal or luminal application of EGF (3-100 riM) produced a sustained increase in I~ and PD in ileal sheets pretreated with [DTAla2,Glu4]deltorphin, but not in tissues treated with U50,488H, NPY, S-14 or NE; a weak, transient increase in I,o was seen in tissues pretreated with DAMGO. The effect of EGF in [D-Ala2,Glu4]deltorphin -treated ileum was concentration-related with As0 (95% C.L.) of 8.1 (5.3-11.3) nM and 9.8 (6.9-12.2) nM for serosal and luminal application, respectively. The activities of luminal and serosal EGF were attenuated by pretreatment of tissues with tetrodotoxin (0.1 gM), an inhibitor of neural conductance. EGF activities were also suppressed by replacement of el-, but not HCO3"from buffers. Pretreatment oftissues with tyrphostin-25 (1 mM), an inhibitor of TK, inhibited the activity of both serosal and luminal EGF, while genistein (0.03-0.2 mM), also an inhibitorof TK, inhibited the activity of luminal EGF, but not that ofserosal EGF. Conclusion: Serosal or luminal EGF regulates ion transport in mouse ileum in which 6 opioid receptors have been activated. These EGF activities were TK-depandent, however, differential effects of genistein suggest that luminal and serosal EGF may involve different kinase cascades.
NITRIC OXIDE-MEDIATED VASCULAR HYPER-RESPONSlVENESS TO ENDOTHELIN. E. M. Redmond, R. Hodges, S. Zhang, P.A. Cahill and J. V. Sitzmann. Johns Hopkins Medical Institutions, Baltimore, MD 21287. Endothelin (ET) is a potent and long lasting vasoconstrictor peptide produced by vascular endothelial cells (1). Recently, we have reported that chronic exposure of cultured rat vascular smooth muscle cells (RVSMC) to NO generating agents caused a significant increase in the number of ET receptors in a time- and dose-dependent fashion (2). To assess the possible functional consequences of this upregulation, we examined the effect of a n NO generating drug, S-nitroso-N-acetylpenicillamine (SNAP) on isolated vessel reactivity. The superior mesenteric artery (SMA) end thoracic aorta (TA) of male Sprague-Dawley rats were removed, cut into 5mm rings and placed in a tissue bath of Krebs solution at 37 °C. Vessel rings, following deactivation of endothelium by one min exposure to 0.3% sodium deoxycholate, were incubated with or without SNAP (250 ruM) for a 5 hr period. Cumulative doseresponse curves to ET-1 (10 1° - 107M) were then obtained, and the ED50 (concentration required to cause 50% of maximum contraction) and Rm~ (maximum force of contraction) were calculated. Data is expressed as force of contraction (g) and is the mean + SEM of 3-4 separate experiments.
] l ,-
. . . .
1.
.
.
.
.
koI [ET~I 1. M
.
L,m l,T-,I,t
In both TA and s M A vessels SNAP pretreatment caused a significant increase in the maximal force of contraction (Rmax) to ET. We conclude that this NO-mediated vascular hyper-responsiveness may in part be due to a marked upregulation of ET-A receptors. (1) Yanagisawe et el., Nature, 332:411-415 (2) Redmond et at., Hepatology (1994) 20:99 A 10.
AGA A B S T R A C T S
PHOSPHOLIPASE Ao,ALONE A~kUJASSOCEATED NITH CALCI~;I, EXPLOSIVELY ACTTVATES CARBONIC AM~YDRASE If(CA II), WHILE INDOb~THACIN(IMC) ANTAGONIZES THIS EFFECT. l°Pu~ca~,Maroela Colt~u,Gabriela Domu~a,Center for Medical Research and Assistance,Simleu Silvaniei, ROMANIA. Considering the importance of phospholipase A~, a cytosolic enzyme wlth a central role in the formation of arachidonic acid,and the role of CK II in the modulation of intracellular pH,we s~udied the effect of phospholipase A~on CA.We also followed the effect on CA I and CA~II induced by associ ation of calcium and IMC to phospholipase A2o METHOD:we followed in vitro the effect on purified ~ n d CA II induced by phospholipase Agand by e quimolar association of:phospholipase A~+Ua;phospholipase Ag+INC;phospholipase A2+Ca+IMC,Assessmen ts were perTormed according to dose-re~oonse relationship,at concentrations between lO- LIO-5M°CA activity was determined by a stopped-flow method (lihalifah RG~1971). RESULTS: Donc° Phospho" Phosphol~- Phosp'ho- Phospholi(M) lipase A 2 pase A2+Ca lipase A 2 pase A2+ +IMC +Ca+IMC IO-~ 26% 4'5% 12% 14% lO-~ 83% 137% 20% 23% lO -6 117% 198% 29% 31% 10-5 180% 300% 33% 35% DISOUSSlON:phospholipase A 2 strongly activates CA II;association of Ca induces an explosive activation of CA II,while CA I activity remains unmodifi ed. The results suggest that the lowering of intracellular pH induced by cytosolic CA II activation could favour the synthesis by phospholipase A.o of arachidonic acid,precursor of prostaglandins.~ntagonization of these activating effects by IMC suggests their acting upon on the common situs of CA II. Thus IMC seems to inhibit not only cyclooxygena se and prostaglandin synthesis,but also phospholipase A 2 and the synthesis of arachidonic acid,
• SUPPRESSION OF VASOACT1VE INTESTINAL PEPTIDE (VIP)INDUCED INCREASES IN ILEAL ION TRANSPORT BY SIGMA LIGANDS. R.K. Rao, P.J.M. Riviere, X. Pascaud, J.L. Junien and F. Porreca. Department of Pharmacology, University of Arizona, Tucson, Arizona, U.S.A and Institut de Recherche Jouveinal, France. Our recent studies have shown that JO 1784 (a o receptor agonist) and neuropeptide Y (NPY) produce net proabsorptive/antisecretory effects in mouse ileum. As most of diarrheas are mediated by hypersecretion of prosecretory agents such as prostaglandinsand VIP, we tested the effects of JO 1784 and NPY on VIP-induced changes in mouse ileal ion transport. Methods: Ion-transport was studied by measuring short circuit current (I,~) and potential difference (PD) across mouse ileum mounted in Ussing chambers in vitro. I,o and PD were recorded before and after serosal application of cumulative doses of VIP. JO 1784 or NPY mediated inhibition and reversal of VIP-induced increase of I,~ were evaluated. Involvement of VIP receptors was determined by evaluating the effects ofVIP-antagonists (VIPA-1 and VIPA-2)] on VIP, JO 1784 and NPY activities. Results: Serosal application of VIP (1-100 riM) produced sustained increase in basal Isc without altering tissue conductance; this activity was concentration-related, with As0 (95% C.L.) of 0.8 (0.3-1:1) nM. Pretreatment of tissues with JO 1784 (10 gM) or NPY (10 nM) (which produced sustained decrease in basal I~) blocked the VIP-induced increase in Isc, resulting in a respective rightward displacement in VIP concentrationresponse curve of 44 or 13 folds. JO 1784 and NPY were also reversed the effect of VIP (10 nM) in a concentration-related manner. Serosal VIPA-1 or VIPA-2 (both 3 gM) produced a slight decrease (15-19%) in basal I,o and significantly reduced the effects of VIP (10 nM) and JO 1784 (10 p.M) without altering the actions of NPY. Conclusion: The findings that VIP antagonists can inhibit the action of JO 1784 suggest that o receptor mediated regulation of ion transport in mouse ileum may involve suppression of tonically released VIP. Thus, o-agonists may prove useful in controlling diarrheas caused by VIP hypersecretion. The reduced potency of NPY in blocking VIP activity, and its insensitivity to V1P antagonists suggest that NPY and JO 1784 may involve different types of receptors.
GASTROENTEROLOGY, Voh 1 0 8 , NO. 4
• EPIDERMAL GROWTH FACTOR (EGF) REVERSES OPIOID DELTA, BUT NOT OPIOID MU OR KAPPA, RECEPTOR-MEDIATED CHANGES IN ILEAL ION TRANSPORT BY A TYROSINE KINASE (TK)-DEPENDENT MECHANISM. ILK. Rao and F. Porreca. Department of Pharmacology, University of Arizona, Tucson, AZ 85724. Our previous studies have shown that EGF produced an increase in shortcircuit current (I~) in mouse ileum in which ganglionic activity was suppressed by ganglionic blockade. As opioids act at a pre-ganglionic site to regulate intestinal ion transport, the activity of EGF was tested in tissues pretreated with opioids with selectivity for 6 ([D-Ala2,Glu4]deltorphin), p. (DAMGO) and K (U50,488H) receptors. EGF activity was also tested in tissues treated with other antisecretory/proabsorptive agents, such as neuropeptide Y (NPY) somatostatin (S-14) or norepinephrine (NE). Methods: Ion-transport was studied by measuring the I,~ and potential difference (PD) across mouse ileal sheets mounted in Ussing chambers in vitro. Effects oflumir/ally or serosally applied mouse EGF on Is~ and PD were evaluated in tissues pretreated with serosal [D-Ala2,Glu4]deltorphin (100 nM), DAMGO (1 gM), U50,488H (3 p.M), NPY (30 nM), S-14 (1 gM) or NE (1 gM); all these compounds produced sustained decrease in ileal I~. Results: Serosal or luminal application of EGF (3-100 riM) produced a sustained increase in I~ and PD in ileal sheets pretreated with [DTAla2,Glu4]deltorphin, but not in tissues treated with U50,488H, NPY, S-14 or NE; a weak, transient increase in I,o was seen in tissues pretreated with DAMGO. The effect of EGF in [D-Ala2,Glu4]deltorphin -treated ileum was concentration-related with As0 (95% C.L.) of 8.1 (5.3-11.3) nM and 9.8 (6.9-12.2) nM for serosal and luminal application, respectively. The activities of luminal and serosal EGF were attenuated by pretreatment of tissues with tetrodotoxin (0.1 gM), an inhibitor of neural conductance. EGF activities were also suppressed by replacement of el-, but not HCO3"from buffers. Pretreatment oftissues with tyrphostin-25 (1 mM), an inhibitor of TK, inhibited the activity of both serosal and luminal EGF, while genistein (0.03-0.2 mM), also an inhibitorof TK, inhibited the activity of luminal EGF, but not that ofserosal EGF. Conclusion: Serosal or luminal EGF regulates ion transport in mouse ileum in which 6 opioid receptors have been activated. These EGF activities were TK-depandent, however, differential effects of genistein suggest that luminal and serosal EGF may involve different kinase cascades.
NITRIC OXIDE-MEDIATED VASCULAR HYPER-RESPONSlVENESS TO ENDOTHELIN. E. M. Redmond, R. Hodges, S. Zhang, P.A. Cahill and J. V. Sitzmann. Johns Hopkins Medical Institutions, Baltimore, MD 21287. Endothelin (ET) is a potent and long lasting vasoconstrictor peptide produced by vascular endothelial cells (1). Recently, we have reported that chronic exposure of cultured rat vascular smooth muscle cells (RVSMC) to NO generating agents caused a significant increase in the number of ET receptors in a time- and dose-dependent fashion (2). To assess the possible functional consequences of this upregulation, we examined the effect of a n NO generating drug, S-nitroso-N-acetylpenicillamine (SNAP) on isolated vessel reactivity. The superior mesenteric artery (SMA) end thoracic aorta (TA) of male Sprague-Dawley rats were removed, cut into 5mm rings and placed in a tissue bath of Krebs solution at 37 °C. Vessel rings, following deactivation of endothelium by one min exposure to 0.3% sodium deoxycholate, were incubated with or without SNAP (250 ruM) for a 5 hr period. Cumulative doseresponse curves to ET-1 (10 1° - 107M) were then obtained, and the ED50 (concentration required to cause 50% of maximum contraction) and Rm~ (maximum force of contraction) were calculated. Data is expressed as force of contraction (g) and is the mean + SEM of 3-4 separate experiments.
] l ,-
. . . .
1.
.
.
.
.
koI [ET~I 1. M
.
L,m l,T-,I,t
In both TA and s M A vessels SNAP pretreatment caused a significant increase in the maximal force of contraction (Rmax) to ET. We conclude that this NO-mediated vascular hyper-responsiveness may in part be due to a marked upregulation of ET-A receptors. (1) Yanagisawe et el., Nature, 332:411-415 (2) Redmond et at., Hepatology (1994) 20:99 A 10.