Human recombinant hemoglobin (rHb1.1) attenuates the inhibitory effects of vasoactive intestinal polypeptide (VIP) and NANC nerve stimulation on the opossum internal anal sphincter (IAS)

A316

AGA ABSTRACTS

GASTROENTEROLOGY,Vol. 108, No. 4

ENDOTHELIAL CELL DERIVED XANTHINE OXIIX)REDUCTASE(XOR) FUNCTIONS AS A SIGNAL TRANSDUCER FOR NEUTROPH]L ADHERENCE TO ENDOTHELIAL CELLS IN VITRO~ U Rang~, EH Smith, ML Blardone, K OcoaneU,GB Bulldey. Dept of Surgery,johns Hepkins Medical Institutions, Baltimore bAD The role ofxanthine oxidoreductase (XOR) in postisehemic reperfusion injury has been characterized by Granger et alI as a two-step process in which xanthine dehydrogenase(XD) is proteolytlcallyeouvertedto xsuthine oxidase (X(3) followed by consequentsuperoxideaniou-mediatedneulrephil adherence to the eodothelium. Studies by Fried at al2 have suggested that other inflammatorymediators such as TNFet and C5A also mediate conversion OfXD to XO in endothelial cells. We soughtto investigatethe role of this trigger mechanism as a fundamental mediator ofinfl~nmafienmeasured by the adherence of fluorescently labeled nentrophtls to endothelial monolayers in vitro. Murine endothelial cell monolayers stimulated with TNT~ for 4 h, washed, and subsequently eoineubated with rat nsutrophils, showed a significant increase in neutrophil adherence over that of controls.

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1

This response was blocked by 80% +/- 10% with prior allopUrinoltreatment thus suggestingthat endothelial derived XOR was necessmy for neutrophil adherence. Furthermore, soybean trypsin inhibitor, a seriHeprotease inhibitor, blocked both proteolytie XD to XO conversion and neutrephil adherence by 80%, suggesting that the XD to XO trigger meehmtism may be a necessary step in this cytokine~ mediatednentrephilresponse. These data suggest that the activated endothelial cell can independentlymediate cytoldne stimulated neutrophil adherence and that XD to XO conversion can be a necessary and proximate transducer of this response.

HUMAN RECOMBINANT HEMOGLOBIN (rHbL 1) ATTENUATES THE INHIBITORY EFFECTS OF VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) AND NANC NERVE STIMULATION ON THE OPOSSUM INTERNAL ANAL SPHINCTER (IAS). S. Rattan. G. Rosenthal, S. Chakder. Dept. of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA and Somatogen, Inc., Boulder, CO. B a c k g r o u n d : Nitric oxide (NO) plays a significant role in the nonadrefiergic noncholinergic (NANC) nerve-mediated gastrointestinal smooth muscle relaxation. Hemoglobin is known to avidly bind NO at the extracelinlar level and inhibit the NANC nerve-mediated smooth muscle relaxation. Recently, a human recombinant hemoglobin (rHb 1.1) has been developed as a potent and stable analog of oxyhemoglobin as a blood substitute. Methods: The IAS circular smooth muscle strips were used f o r the recording of the isometric tension. The effects of NANC nerve stimulation by electrical field stimulation (EFS), NO, VIP, peptide histidine isoleucine (PHI) and isoproterenol on the basal tone of the IAS were examined before and after different concentrations of rHbl.L Results: EFS caused frequency-dependent relaxation of the IAS. NO, VIP, PHI and isoproterenol caused concentration-dependent fall in the basal tone of the IAS. rHbl.1 (3 x l 0 "6 M) caused complete obliteration of IAS relaxation by different concentrations of NO. The henm_ob~lobincaused significant and concentration-dependent (3 xl0 "6 to 3 xl0- M) attenuation of EFS-induced IAS relaxation. Interestingly, rHbl.1 also caused suppression of the VIP-induced fall in the basal tension of the I.AS. In control experiments, the fall in IAS tension by 1 x 10-7, 3 x 10"7, 1 x 10-6 and 3 x 10-6 M VIP were 39.2 +11,8, 70.4+ 6.3, 82.2 + 5.8 and 85.1+ 5.1% respectively. The hemoglobin (3 x 105 M) attenuated these responses to 14.0 + 8.3, 38.5 :i: 12.6, 42.6 + 6.9 and 58.9 + 5.6 % respectively (p < 0.05; n --5). However, fall in the IAS tension by PHI and isoproterenol were not modified by rHbl.1 (p > 0.5; n =5). The data suggest that in the IAS, NO-dependent actions of EFS and VIP are at the presynaptic site and I-Ib acts at the neuromuscular junction between the NANC nerves and the smooth muscle cells. Conclusion: In the IAS, a part of action of VIP may be via NO release from the sources other than the effeetor site smooth muscle ceils. Furthermore, the data suggest that in addition to its role as an inhibitory neurotransmitter, NO serves as a mediator in the IAS.

THE FUNDAMENTAL HEMODYNAMIC MECHANISM ' OF GALLBLADDERISCHEMIA DUR/NG CARDIOGENIC SHOCK, PM Rellly, TJ Toung, M Miyachi, HI Schiller, GB Bulkley. Dept of Surgery, Johns Hopkins Medical Institutions, Baltimore MD

• BUTYl)ATE IS CYTOPROTECTIVE AGAINST OXIDANT INJURY IN INTESTINAL EPITHELIAL CELLS (IEC-18). $oel Rctskv. Glendon Burress, Mark Musch, May Ciancio, Eugene Chang. Section o f ~terology, Department of Medicine, University of Chicago, Chicago, IL

Puruose: We qualitatively and quantitatively evaluated the hemodynamic response of the gallbladder to eardiogenic shock in pigs. Methods:Progressive, graded, steady-stateincreasesin Pericardial pressure from 0-8mmI-Ig (tamponade) resulted in corresponding decreases in cardiac output (CO), arterial pressure (PA) and an increase in total peripheral resistance (TPR), thereby simulating precisely the hemodynamics of cardiogenic shock. Gallbladder blood flow (QoB) was measured with 15-1-1/zm radiolabelled microspheres, sonicated and injected into the left atrium under steady-state conditiom at baseline (EL) and maximal tamponade (MAX) by the reference organ technique. Gallbladder vascular resistance (Rae) was calculated:

Butyrate is a major enterocy~ nutrient and is avidly absorbed by colonic and small intestinal epithelial cells. Recent evidence suggests that butyrate is effective in healing inflamed mucosu of diversion cofitis, ulcerative colitis and ponchitis however, the mechanism is unknown. We studied the cytoprotective efficacy of butyrate against oxidant injmy which is likely a major mechanism of tissue injury in inflammation. In addition, since heat shock proteins (lISP) are cytoprotective against oxidant injury in IEC-18 cells, we evaluated the effect of butyrate on lISP synthesis as a potential mechanism of the butyrate effect. Methods: IEC-! 8 cells were grown in tissue culture at 370C. Cell caltures were incubated with butyrate at concentrations of 0, 1, 3, 10 and 3(hnM for 2 hours. Cytoproteotion assays were performed by measuring Crsl release after subjecting the butyrate treated cells to 0, .03,.1, .3, 1 and 3ram monochioramine (a strong oxidant.) lISP synthesis was determined by Western blotting using MAb C92 (StressGen) against HSP-72 extracted from the butyrate treated cells. To determine whether or not butyrate angneats tim heat shock response, cells treated with and without butyrate were heat shocked at 38, 39, 40, 41 and 42°C for 23 minutes then returned to 37°C for 2 hours prior to extraction of proteins. Results: After exposure to lmM monochloramine, cells incubated with 10ram butyrate released 4 I% less Crsj than control. This effect, although less pronounced, was also seen with 0.3 and 3mM monochloramine. However, HSP-72 synthesis was not induced by butyrate at all concentrations tested as compared with contr01. Varyin~gthe degree of confluency, butyrate incubation time, and the nutrient content of the media had no effect on this result. Treatment with butyrate diminished the production of liSP-72 that is normally seen after heat shocking at38, 39, 40 and 41°C. This effect was most significant at 41°C where 10mM butyrate reduced HSP-72 synthesis by 55%. Condualuns: Bulyrate is cytoprotective against oxidant injury in intestinal epithelial cells which may explain its efficacy in healing inflamed macosa. However, the mechanism of this effect remains unclear as butyrate does not appear to induce heat shock protein synthesis. In addition, b u t y r a t e , ~ s rather than augments the normal heat shock response seen at 38-41°C as measured by production of HSP-72.

SHOCK ALONE (nffi14) CO ~ TPR Q~ (ml/min/'100g) (nmlHglnd/min/100g) (ml/min/100S)

R~

(mmHglmllmlnl!OOg)

BL

14.7+,7

6.2+.2

50+5

2.14-.2

MAX

6.14-.3

9.2+.4

i5+2

5.14-.8

%BL

42:~2

146:1:6 304-3* 240+20* * p < .05 vs %BL CO; i p < .05 vs •BL TPR

Shock caused disproportionate gallbladder ischemia due largely to selective splanchnicvasoconstriction.Confirmed a-adrenergic blockade (a-X) was induead with phenoxybenzamine,vasopressiLblockade (VP-X) with Manning compound, and renin-angiotensin axis bloclc 'e (ACE-X) with enalaprll: PHARMACOLOGIC ANTAGONISTS n

Qm (%BL)

~-X + SHOCK

9

33+5

R ~ (%BL) 300±70

VP-X + SHOCK

11

274-4

4004-100

ACE-X + SHOCK

13

44+4*

1604-20"

*p<.05 vs SHOCK ALONE .

Only enalapril blocked the disproportionate vasocoasU'iction and thereby anenuated the isehemia in the gallbladder. Conclusions Disproportionate ischemia of the gallbladder during cardiogenic shock is due primarilyto selective gallbladder vasospasm mediated largely by the renin-angiotemin axis.