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Release of Vasoactive Intestinal Polypeptide (VIP) by Electric Stimulation of the Vagal Nerves
72:373-375, 1977 Copyright © 1977 by The Williams & Wilkins Co.
Vol. 72, No.2 Printed in U.s A.
GASTROENTEROLOGY
RAPID COMMUNICATION O.B. Schaffalitzky de Muckadell, J. Fahrenkrug, and J.J. Holst. RELEASE OF VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) BY ELECTRIC STIMULATION OF THE VAGAL NERVES. Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark. ABSTRACT. The concentration of immunoreactive VIP was measured in portal venous and peripheral arterial plasma in anesthetized pigs. Following electric stimulation of the vagal nerves the median concentration of VIP in portal plasma rose from 21 to 58 pmol 1- 1 and a simultaneous increase was found in arterial plasma. Atropine did not abolish this effect of vagal stimulation. The response is interpreted as increased release of VIP. Vasoactive intestinal polypeptide (VIP) isolated from hog small intestine l is now known to have a wide spectrum of biologic activity2. However, its physiological role and stimulus to release remain to be determined. Recently immunocytochemical and radioimmunochemical studies have demonstrated VIP in central and peripheral neurons 3 - 6 , suggesting a neurotransmitter function of the peptide. The present study was undertaken to investigate the effect of vagal stimulation on the release of VIP.
A laparotomy was performed, the pylorus was ligated, and gastric content was continuously drained by an orogastric tube. In three of the pigs the splancnic nerves were cut bilaterally. A thoracotomy was then performed, the vagal trunks identified above the diaphragm and cut below the heart. The peripheral cut ends of the nerves were passed through a bipolar platinum electrode 7 • The vagal nerves were stimulated with constant current square-wave impulses (impulse duration 5 msec, 20 Hz, 10 mAmp) for five min (delivered by an Electronic Square-wave Stimulator, Palmer, U.K.). Applied voltage and current was monitored on a Dual-beam Oscilloscope, type 502 A (Tektronix, Guernsey Ltd., U.K.). In three of the pigs stimulation was repeated 30 min after intravenous injection of 0.5 mg atropine per kg. Blood samples were drawn from the portal vein 15, 10, 5, and 0 min before and 0.5, 1, 3, 5, 10, 15, and 30 min after the start of the stimulation. In three of the pigs samples were simultaneously drawn from the femoral artery. Blood was collected in ice-chilled tubes c~nfaining 500 k.I.U. aprotinin (Trasylol , Bayer, Leverkusen, GFR) and 50 I.U. heparin per ml blood. Samples were centrifuged at 4 °c and plasma stored at -20 °c until assayed. The concentration of VIP in plasma was measured radioimmunochemically8. Antibodies were raised in rabbits against highly purified natural porcine VIP (kindly donated by Prof. V. Mutt, Karolinska Institutet, Stockholm, Sweden) covalently coupled to bovine serum albumin. The antiserum used (5603-6) reacted with an effective equilibrium constant of 3.5 lOll 1 mol- l and no crossreactivity was found with porcine gastric inhibitory
METHODS Eight anesthetized female pigs of Danish landrace (weight 17-25 kg, age 8-12 weeks) were investigated. Food but not water was withdrawn 18 h before experiments. (R) Azaperone (Sedaperone ) 4 mg per kg intramuscularly was used for premedication. Anesthesia was induced with halothane/02/N20 and maintained with chloralose (Merck, Darmstadt, GFR), 70 mg per kg intravenoust*r Polyethylene catheters (Argyle feeding tube 8 CH Sherwood Medical Industry Inc., St. Louis, Missouri, U.S.A.) were inserted into the portal vein and left femoral artery and vein. Isotonic saline was infused into the femoral vein throughout the experiment. Intraarterial blood pressure and rectal temperature were continuously monitored (EMT 34 transducer, Mingograph, E. Sh6nander, Stockholm, Sweden, and Temperature recorder, type Z8, Ellab, Copenhagen, Denmark). Submitted August 4, 1976 Address reprint requests to Dr. Schaffalitzky de Muckadell 373
peptide, porcine pancreatic glucagon, porcine enteroglucagon, human pancreatic polypeptide, synthetic bovine substance P, porcine natural secretin, or synthetic ovine somatostatin in concentrations below 105 pmol 1- 1 • 125 1 _ VIP was prepared by a chloramine T method to a specific radioactivity of approximately 900 ~Ci per nmol hormone. Highly purified natural porcine VIP was used as standard and dissolved in hormone-free plasma prepared from each pig by charcoaling. Antibody-bound and free label were separated by absorption to plasma-coated charcoal. The lowest VIP concentration to be distinguished from zero with 95 per cent confidence was 3.3 pmol 1- 1 • The within and between assay reproducibility expressed as coefficient of variation was 0.07 and 0.15 at a level of 44 pmol 1- 1 • All samples were assayed in triplicate. Statistical analysis were performed using the Wilcoxon matched-pairs signed-ranks test or the Friedman two-way analysis of variance 9 • Differences with P values of less than 0.05 were considered significant. RESULTS Median portal concentration of VIP in the basal state was 21 pmol 1- 1 with an interquartile range of 12-33 pmoll- 1 (Fig. lA). Within a few sec after beginning the stimulation, the small bowel and stomach showed increased active peristalsis which continued throughout the period of stimulation. The median concentration of VIP in portal plasma rose significantly within 0.5 min and reached a peak value of 58 pmol 1- 1 after three min (Fig. lA). Within ten min after cessation of the stimulation the concentration decreased to the prestimulated values. The changes in the concentration of VIP in portal plasma were accompanied by a simultaneous significant increase (range 11-33 pmol 1- 1 ) following vagal stimulation in the concentration of VIP in arterial plasma (Fig. lB). The response was not abolished by atropine (Fig. lC). The basal and stimulated concentrations of VIP in plasma were significantly higher in the three pigs with cut splancnic nerves. In the basal state concentrations of VIP ranged from 33 to 101 pmol 1- 1 in pigs with cut splancnic nerves and from 8 to 31 pmol 1- 1 when splancnic
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Fig. 1. Effect of electric stimulation of the vagal nerves on the median concentrattion of VIP in plasma of anesthetized pigs. Horizontal bars indicate the duration of the stimulation. A: Portal plasma, median and interquartile range (hatched area), n = 8. B: Portal plasma (PV) and femoral arterial plasma (FA), n = 3. C: Portal plasma. - before and + after 0.5 mg atropine per kg given intravenously, n = 3.
nerves were intact. During stimulation the range in concentration of VIP was 72-159 and 20-60 pmol 1- 1 respectively. Systemic arterial blood pressure was depressed less than 7 mmHg in five pigs and not affected in three pigs. DISCUSSION The present study has shown that electric stimulation of the vagal nerves causes an increase in the concentration of VIP in portal and peri374
pheral plasma and that this action is atropine-resistant. The response is believed to reflect increased release of VIP into the circulation since increased concentrations were noted also in peripheral arterial blood. Electric stimulation of the peripheral cut end of the vagus nerve does not modify superior mesenteric blood flow 10 , and there is no evidence that the vagal nerves carry any parasympathetic vasodilator fibers to the vessels of the small intestine, but subdiaphragmatic activation of high threshold fibers are accompanied by a decrease in intestinal blood flow and an increase in systemic blood pressure 11 • To elucidate this further portal blood flow was measured electromagnetically (Biotronex BL-610 pulsed-logic flowmeter, BL 5060-056 flow transducer) in one pig, and an increase from 320 ml min- 1 to 445 ml min- 1 was seen during vagal stimulation. The observed release of VIP is considered to be a direct consequence of vagal stimulation but it can not be ruled out that a physiologic response to vagal stimulation (peristalsis for example) resulted in VIP release. VIP is a strong agonist for pancreatic bicarbonate secreiton 12 • The atropine-resistant VIP response to vagal stimulation might therefore be involved in the atropine-resistant action of the vagus nerves on pancreatic blood flow and pancreatic exocrine secretion 13 • Furthermore, since the concentration of VIP in arterial plasma increased during stimulation, VIP in addition to a local (paracrine) effect might exert hormonal control of other metabolic events 2
REFERENCES 1. Said SI, Mutt V: Polypeptide with broad biological activity: Isolation from small intestine. Science 169: 1217-1218, 1970. 2. Said SI: Vasoactive intestinal polypeptide (VIP): Current status. In Thompson JC (ed) Gastrointestinal Hormones. University of Texas Press. Austin and London 1975. 3. Bryant MG, Polak JM, Modlin I et al: Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet 1:991-993, 1976. 4. Larsson L-I, Edvinson L, Fahrenkrug J et al: Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves. Brain Research 113:400-404, 1976. 5. Larsson L-I, Fahrenkrug J, Schaffalitzky de Muckadell OB et al: Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc Nat Acad Sci USA 73:3197-3200, 1976. 6. Said SI, Rosenberg NR: Vasoactive intestinal Polypeptide: Abundant Immunoreactivity in Neural Cell Lines and Normal Nervous Tissue. Scinece 192:907-908, 1976. 7. Daniel PM, Henderson JR: The effect of vagal stimulation on plasma insulin and glucose levels in the baboon. J Physiol 192:317-327, 1967. 8. Fahrenkrug J, Schaffalitzky de Muckadell OB: Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. Submitted for publication. 9. Siegel S: Non parametric statistics for the behavioral sciences. McGraw-Hill, New York, 1956. 10. Fara JW, Rubinstein EH, Sonnenschein RR: Intestinal hormones in mesenteric vasodilation after intraduocenal agents. Am J Physiol 223: 1058-1067, 1972. 11. Kewenter J: The vagal control of the jejunal and ileal mobility and blood flow. Acta Physiol Scand 65: suppl. 251, 1965. 12. Konturek SJ, Pucher A, Radecki T: Comparison of vasointestinal peptide and secretin in stimulation of pancreatic secretion. J Physiol 225:497509, 1976. 13. Hickson JCD: The secretory and vascular response to nervous and hormonal stimulation in the pancreas of the pig. J Physiol 206:299-322, 1970.
Acknowledgements: The skilful technical assistance of Anita Hansen, Lene Poulsen, Nina Rasmussen and Anne Sylvest is gratefully acknowledged. The study was supported by grants from the Danish Medical Research Foundation (J.nr. 512-4154, 512-5676), The Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faroe Islands, and Greenland (J.nr. 75/76-40), and Kong Christian den X's Fond. 375
Vol. 72, No.2 Printed in U.s A.
GASTROENTEROLOGY
RAPID COMMUNICATION O.B. Schaffalitzky de Muckadell, J. Fahrenkrug, and J.J. Holst. RELEASE OF VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) BY ELECTRIC STIMULATION OF THE VAGAL NERVES. Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark. ABSTRACT. The concentration of immunoreactive VIP was measured in portal venous and peripheral arterial plasma in anesthetized pigs. Following electric stimulation of the vagal nerves the median concentration of VIP in portal plasma rose from 21 to 58 pmol 1- 1 and a simultaneous increase was found in arterial plasma. Atropine did not abolish this effect of vagal stimulation. The response is interpreted as increased release of VIP. Vasoactive intestinal polypeptide (VIP) isolated from hog small intestine l is now known to have a wide spectrum of biologic activity2. However, its physiological role and stimulus to release remain to be determined. Recently immunocytochemical and radioimmunochemical studies have demonstrated VIP in central and peripheral neurons 3 - 6 , suggesting a neurotransmitter function of the peptide. The present study was undertaken to investigate the effect of vagal stimulation on the release of VIP.
A laparotomy was performed, the pylorus was ligated, and gastric content was continuously drained by an orogastric tube. In three of the pigs the splancnic nerves were cut bilaterally. A thoracotomy was then performed, the vagal trunks identified above the diaphragm and cut below the heart. The peripheral cut ends of the nerves were passed through a bipolar platinum electrode 7 • The vagal nerves were stimulated with constant current square-wave impulses (impulse duration 5 msec, 20 Hz, 10 mAmp) for five min (delivered by an Electronic Square-wave Stimulator, Palmer, U.K.). Applied voltage and current was monitored on a Dual-beam Oscilloscope, type 502 A (Tektronix, Guernsey Ltd., U.K.). In three of the pigs stimulation was repeated 30 min after intravenous injection of 0.5 mg atropine per kg. Blood samples were drawn from the portal vein 15, 10, 5, and 0 min before and 0.5, 1, 3, 5, 10, 15, and 30 min after the start of the stimulation. In three of the pigs samples were simultaneously drawn from the femoral artery. Blood was collected in ice-chilled tubes c~nfaining 500 k.I.U. aprotinin (Trasylol , Bayer, Leverkusen, GFR) and 50 I.U. heparin per ml blood. Samples were centrifuged at 4 °c and plasma stored at -20 °c until assayed. The concentration of VIP in plasma was measured radioimmunochemically8. Antibodies were raised in rabbits against highly purified natural porcine VIP (kindly donated by Prof. V. Mutt, Karolinska Institutet, Stockholm, Sweden) covalently coupled to bovine serum albumin. The antiserum used (5603-6) reacted with an effective equilibrium constant of 3.5 lOll 1 mol- l and no crossreactivity was found with porcine gastric inhibitory
METHODS Eight anesthetized female pigs of Danish landrace (weight 17-25 kg, age 8-12 weeks) were investigated. Food but not water was withdrawn 18 h before experiments. (R) Azaperone (Sedaperone ) 4 mg per kg intramuscularly was used for premedication. Anesthesia was induced with halothane/02/N20 and maintained with chloralose (Merck, Darmstadt, GFR), 70 mg per kg intravenoust*r Polyethylene catheters (Argyle feeding tube 8 CH Sherwood Medical Industry Inc., St. Louis, Missouri, U.S.A.) were inserted into the portal vein and left femoral artery and vein. Isotonic saline was infused into the femoral vein throughout the experiment. Intraarterial blood pressure and rectal temperature were continuously monitored (EMT 34 transducer, Mingograph, E. Sh6nander, Stockholm, Sweden, and Temperature recorder, type Z8, Ellab, Copenhagen, Denmark). Submitted August 4, 1976 Address reprint requests to Dr. Schaffalitzky de Muckadell 373
peptide, porcine pancreatic glucagon, porcine enteroglucagon, human pancreatic polypeptide, synthetic bovine substance P, porcine natural secretin, or synthetic ovine somatostatin in concentrations below 105 pmol 1- 1 • 125 1 _ VIP was prepared by a chloramine T method to a specific radioactivity of approximately 900 ~Ci per nmol hormone. Highly purified natural porcine VIP was used as standard and dissolved in hormone-free plasma prepared from each pig by charcoaling. Antibody-bound and free label were separated by absorption to plasma-coated charcoal. The lowest VIP concentration to be distinguished from zero with 95 per cent confidence was 3.3 pmol 1- 1 • The within and between assay reproducibility expressed as coefficient of variation was 0.07 and 0.15 at a level of 44 pmol 1- 1 • All samples were assayed in triplicate. Statistical analysis were performed using the Wilcoxon matched-pairs signed-ranks test or the Friedman two-way analysis of variance 9 • Differences with P values of less than 0.05 were considered significant. RESULTS Median portal concentration of VIP in the basal state was 21 pmol 1- 1 with an interquartile range of 12-33 pmoll- 1 (Fig. lA). Within a few sec after beginning the stimulation, the small bowel and stomach showed increased active peristalsis which continued throughout the period of stimulation. The median concentration of VIP in portal plasma rose significantly within 0.5 min and reached a peak value of 58 pmol 1- 1 after three min (Fig. lA). Within ten min after cessation of the stimulation the concentration decreased to the prestimulated values. The changes in the concentration of VIP in portal plasma were accompanied by a simultaneous significant increase (range 11-33 pmol 1- 1 ) following vagal stimulation in the concentration of VIP in arterial plasma (Fig. lB). The response was not abolished by atropine (Fig. lC). The basal and stimulated concentrations of VIP in plasma were significantly higher in the three pigs with cut splancnic nerves. In the basal state concentrations of VIP ranged from 33 to 101 pmol 1- 1 in pigs with cut splancnic nerves and from 8 to 31 pmol 1- 1 when splancnic
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x 0
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Fig. 1. Effect of electric stimulation of the vagal nerves on the median concentrattion of VIP in plasma of anesthetized pigs. Horizontal bars indicate the duration of the stimulation. A: Portal plasma, median and interquartile range (hatched area), n = 8. B: Portal plasma (PV) and femoral arterial plasma (FA), n = 3. C: Portal plasma. - before and + after 0.5 mg atropine per kg given intravenously, n = 3.
nerves were intact. During stimulation the range in concentration of VIP was 72-159 and 20-60 pmol 1- 1 respectively. Systemic arterial blood pressure was depressed less than 7 mmHg in five pigs and not affected in three pigs. DISCUSSION The present study has shown that electric stimulation of the vagal nerves causes an increase in the concentration of VIP in portal and peri374
pheral plasma and that this action is atropine-resistant. The response is believed to reflect increased release of VIP into the circulation since increased concentrations were noted also in peripheral arterial blood. Electric stimulation of the peripheral cut end of the vagus nerve does not modify superior mesenteric blood flow 10 , and there is no evidence that the vagal nerves carry any parasympathetic vasodilator fibers to the vessels of the small intestine, but subdiaphragmatic activation of high threshold fibers are accompanied by a decrease in intestinal blood flow and an increase in systemic blood pressure 11 • To elucidate this further portal blood flow was measured electromagnetically (Biotronex BL-610 pulsed-logic flowmeter, BL 5060-056 flow transducer) in one pig, and an increase from 320 ml min- 1 to 445 ml min- 1 was seen during vagal stimulation. The observed release of VIP is considered to be a direct consequence of vagal stimulation but it can not be ruled out that a physiologic response to vagal stimulation (peristalsis for example) resulted in VIP release. VIP is a strong agonist for pancreatic bicarbonate secreiton 12 • The atropine-resistant VIP response to vagal stimulation might therefore be involved in the atropine-resistant action of the vagus nerves on pancreatic blood flow and pancreatic exocrine secretion 13 • Furthermore, since the concentration of VIP in arterial plasma increased during stimulation, VIP in addition to a local (paracrine) effect might exert hormonal control of other metabolic events 2
REFERENCES 1. Said SI, Mutt V: Polypeptide with broad biological activity: Isolation from small intestine. Science 169: 1217-1218, 1970. 2. Said SI: Vasoactive intestinal polypeptide (VIP): Current status. In Thompson JC (ed) Gastrointestinal Hormones. University of Texas Press. Austin and London 1975. 3. Bryant MG, Polak JM, Modlin I et al: Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet 1:991-993, 1976. 4. Larsson L-I, Edvinson L, Fahrenkrug J et al: Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves. Brain Research 113:400-404, 1976. 5. Larsson L-I, Fahrenkrug J, Schaffalitzky de Muckadell OB et al: Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc Nat Acad Sci USA 73:3197-3200, 1976. 6. Said SI, Rosenberg NR: Vasoactive intestinal Polypeptide: Abundant Immunoreactivity in Neural Cell Lines and Normal Nervous Tissue. Scinece 192:907-908, 1976. 7. Daniel PM, Henderson JR: The effect of vagal stimulation on plasma insulin and glucose levels in the baboon. J Physiol 192:317-327, 1967. 8. Fahrenkrug J, Schaffalitzky de Muckadell OB: Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. Submitted for publication. 9. Siegel S: Non parametric statistics for the behavioral sciences. McGraw-Hill, New York, 1956. 10. Fara JW, Rubinstein EH, Sonnenschein RR: Intestinal hormones in mesenteric vasodilation after intraduocenal agents. Am J Physiol 223: 1058-1067, 1972. 11. Kewenter J: The vagal control of the jejunal and ileal mobility and blood flow. Acta Physiol Scand 65: suppl. 251, 1965. 12. Konturek SJ, Pucher A, Radecki T: Comparison of vasointestinal peptide and secretin in stimulation of pancreatic secretion. J Physiol 225:497509, 1976. 13. Hickson JCD: The secretory and vascular response to nervous and hormonal stimulation in the pancreas of the pig. J Physiol 206:299-322, 1970.
Acknowledgements: The skilful technical assistance of Anita Hansen, Lene Poulsen, Nina Rasmussen and Anne Sylvest is gratefully acknowledged. The study was supported by grants from the Danish Medical Research Foundation (J.nr. 512-4154, 512-5676), The Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faroe Islands, and Greenland (J.nr. 75/76-40), and Kong Christian den X's Fond. 375