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An endogenous peptide that induces long-term blood pressure elevation
Life
AN ENDOGENOUS PEPTIDE THAT INDUCES LONG-TERM BLOOD PRESSURE ELEVATION Gary L. Wright, Stephen Fish, Peter Johnson* and William D. McCumbee Departments of Physiology and Anatomy Barshall WrsiversitySchool of Medicine ttwltinqtan, WV 25704-2901 and *Department of Chemistry Ohio University Athens, DH 45701-2979 (Received in final form May 10, 1988) Summary A peptide was recently isolated from the blood of spontaneously hypertensive (SH) rats that stimulated an increase of calcium uptake by vascular tissue in vitro. In the present study normotensive rats were given nanomolar amounts of this peptide by intravenous or picomolar amounts by intracerebral injection and the effect on blood pressure was recorded. Injection of the peptide into the circulation had no significant effect on the elevation of blood pressure. By comparison, the injection of the compound into the third ventricle of the brain resulted in the elevation of blood> pressure to hypertensive levels. The blood pressure response was characterized by a prolonged period of onset with maximal elevation observed several days after the beginning of treatment. Subsequently, the increase in blood pressure was well maintained with significant elevation noted days following the cessation of treatment. The spontaneously hypertensive rat is a widely studied model of genetically programmed hypertension that provides close facsimiles of essential hypertension in the human. Hypertension in these rats is believed to be due primarily to peripheral vasoconstriction that is linked in some fashion to altered vessel smooth muscle contractility (1). The mechanisms of change in smooth muscle contractility is not certain, but there is some evidence suggesting that abnormal cellular handling of calcium is involved. Contractile dependency and sensitivity to extracellular calcium is increased in blood vessels of SH rats, a phenomenon possibly related to increased calcium permeability of the plasmalennna and reduced microsomal uptake of There is also a large body of calcium in SH rat smooth muscle (2-6). evidence that implicates both the central and peripheral nervous system in the development of hypertension. In the SH rat, the neurogenic component of hypertension may be the result of increased peripheral sympathetic neural activity (7-9). Several lines of evidence suggest that hypothalmic areas of the central nervous system are responsible for this activity which may be aggravated by impaired baroreflexes in the animal (10-12). The changes in vessel contractility and smooth muscle handling of calcium have been linked to the existence of a humoral substance in the SH and other models of hypertension. A number of laboratories have demonstrated 0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
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the presence of a plasma factor that sensitizes the vasculature to pressor agents (13-16), while other groups have reported hypertensive effects of Recently, an renal extracts and serum from hypertension models (17-22). extract of erythrocytes from SH rats was prepared that had a stimulatory effect on calcium uptake by vascular tissue _in vitro and a hypertensive effect when injected into normotensive rats (23,24). The compound having a stimulatory effect on calcium uptake has been isolated and tentatively identified as a small, acidic, calcium-dependent peptide with an amino acid composition of asp/asn (1.41), ser (1.02), glu/gln (1.00) and gly (2.00) (25). Based on the assumption that the peptide contained a single glutamic or glutamine residue, it was calculated that only nanomolar concentrations of materials were required to induce significant stimulation of calcium uptake. Methods In the present study we injected this peptide into 6 week old, male, Wistar Kyoto normotensive rats and observed its effect on blood pressure. The peptide was isolated from the erythrocytes of SH rats as previously The step necessary to completely eliminate phosphate described (25). contamination was deleted to ensure reasonable yields of the peptide. A portion of the sample was analyzed for glutamic/glutamine and phosphate content (25). Based on the assumption that the peptide contains one glu/gln residue, approximately 25 nmoles of the compound in 0.4 mL of distilled water (pH 7.0) was injected by tail vein. A second group of rats received 0.1 nmoles of peptide in 0.01 mL of 0.9% NaCl via a cannula to the third The cannula was implanted stereotaxically and ventricle of the brain. affixed to the skull with dental acrylic and screws. The cannula was sealed off between injections with a threaded cap. Controls received similar volumes of sodium phosphate buffer (pH 7.4). After blood pressure experiments were completed, the rats were given an overdose of nembutal and perfused through the heart with normal saline (150 mL) and 10% buffered formalin (300 mL). With the ventricular cannula left in place, the rostra1 calvaria and a portion of the dental acrylic cannula mount were dissected away. The cerebrum just rostra1 to the cannula was removed, along a frontal plane, so that the position of the cannula tip in the third ventricle could be verified. The brain was then completely removed and inspected for gross abnormalities. Three brains (one control and two experimental animals) were sectioned at 50 urn on a freezing microtome and prepared for microscopic analysis with the thionin stain. Blood pressure and the heart rate of each rat was determined without use of anesthetic using the Marco Programmed Electrosphygmomanometer with tail cuff and pneumatic transducer. The blood pressure was recorded at two day intervals for eight days prior to injections and the last three values were averaged as a preinjection control. Analysis of variance and Student's ttest were used to determine significant differences in the data. Results The mean blood pressure of normotensive rats receiving the peptide by intravenous injection was not significantly different from phosphate control values. In contrast those animals receiving single or multiple intracerebral injections of the peptide exhibited hypertensive elevation of blood pressure (Figs. 1 and 2). The rats given a single injection of peptide showed an average maximal elevation of blood pressure of about 25 torr, achieved at 3
Vol. 43, No. 2, 1988
Blood Pressure and an Endogenous Peptide
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Fig. 1 Systolic blood pressure of Wistar Kyoto rats that were given a single injection of peptide (dashed line, open circle) or phosphate buffer (solid circle). Each of the peptide-treated group (n=6) received approximately 0.1 nanomole of the peptide in 0.01 mL of 0.9% NaCl, injected into the third ventricle of the brain. Controls (n=5) received a similar volume of phosphate buffer. The hatched area shows the mean + SEM systolic blood pressure of control rats prior to the injection of material. An asterisk or inverted cross indicates that values were significantly different from the preinjection value or phosphate injected control, respectively, p
114
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Gross and microscopic examination of the brains revealed no abnormalities except for minor widening of the ventricles near the cannulae. Discussion These findings suggest that the peptide isolated form the erythrocytes of SH rats, in addition to its potent stimulatory effect on calcium uptake in tissue may cause the elevation of blood pressure to hypertensive levels without a change in cardiac rate when injected into the brains of normotensive rats. The pressor response elicited appears unique in terms of the long
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hY Fig. 2 Systolic blood pressure in Wistar Kyoto rats that were given daily intracerebral injection of peptide (dashed line) or phosphate Following the determination of preinjection buffer (solid line). control values, Group b rats (n=9) received injections of peptide for a period of 1 days after which phosphate buffer was injected. In a parallel experiment, Group fi rats (n=6) received phosphate buffer injections for 7 days followed by peptide for the remainder Horizontal bars indicate the mean f of the experimental period. As asterisk indicates a SEM of each group prior to injection. significant difference from the preinjection value, p
Vol. 43, No. 2, 1988
Blood Pressure and an Endogenous Peptide
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The interval required for development and the prolonged recovery period. mechanism of the peptide-induced increase in blood pressure may be linked with the compound's influence on tissue metabolism of calcium. However, the discrepancy between the time required for evidence of pressor activity (days) and stimulation of calcium uptake (minutes) renders the relationship unclear. An alternative explanation is that the elevation in blood pressure noted was an artifact of localized damage and CNS lesions leading to hypertension. However, an examination of brains revealed no apparent differences between control and peptide-treated groups. The recovery of peptide injected animals (Fig. 1) and lack of symptoms other than elevation of blood pressure also indicates this explanation is unlikely. The finding that the purified peptide had little effect when given by intravenous injection was surprising in light of previous observations of severe hypertension induced by semipurified preparations given via this route (23,24). One explanation of the apparent loss of activity is that the amount of material injected was below a threshold level for biological action due to peptide degradation, sequestration and/or excretion in the peripheral circulation. Alternately, a carrier molecule or peptide-cofactor may have been lost in the final steps of purification. In any case the administration of the peptide by intracerebral cannula was clearly more effective in inducing blood pressure elevation than intravenous injection of the compound. While the results indicate that the peptide can cause the elevation of blood pressure, the significance of the compound to hypertension in the SH rat is less clear. The peptide has been shown to be present in normotensive animals. However, the yield of the compound from erythrocytes of SH rats is consistently 2 to 5 fold greater than from normotensive rats (23). Dietary manipulation which attenuates blood pressure in SH rats has been found to cause a significant reduction in blood levels of the peptide (26). Normotensive rats subjected to the same dietary regimen, on the other hand, did not show an effect on blood pressure nor in the levels of calcium stimulator activity. Additionally, the blood levels of calcium stimulator activity in 5 week old SH rats, prior to a significant rise in blopd pressure, was not different from that noted in similarly aged normotensive rats (27). Hence, the increase in concentration of the peptide that occurs following maturation and the rise of blood pressure in SH rats, is absent in normotensive rats that do not undergo hypertensive elevation of blood pressure after maturation. Taken as a whole, these observations suggest that the peptide may play a role in the development of high blood pressure in the SH rat. References
:: 3. 4.
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D.F. BOHR, Fed. Proc. 33, 127 (1974). O.L. PEDERSEN, Arch. Int. Pharmacodyn. de Therap. 239, 208 (1979). R.C. BHALLA, R.C. WEBB, D. SINGH, T. ASHLEY and T. BROCK, Molt. Pharmacol. I4, 468 (1978). K. AOKI, Y. YAMASHITA, A. SUZUKI, K. TAKIKAWA and K. HOTTA, Clin. Exptl. Pharmacol. Physiol. 3, 27 (1976). S. SHIBATA, M. KUCHII, and T. TANIGUCHI, Blood Vessels l2, 279 (1975). C.Y. KWAN, Can. J. Physiol. Pharmacol. 63, 366 (1985). W.V. JUDY, A.M. WATANABE and D.P. HENRY, Cir. Res. 38 2 , 21 (1976). A. NAGAOKA and W. LOVENBERG, Life Sci. l9, 29 (1977F . M.F. ROIZEN, V. WEISE and H. GROBECKER, Life Sci. 17(2), 283 (1975).
116
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14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Blood Pressure and an Endogenous Peptide
Vol. 43, No. 2, 1988
J. CIRJELLO, R.L. KLINE, T.X. ZANG and M.M. CAVERSON, Brain Res. 310, 355 (1984). M.C. ANDRESEN, S. KURAOKA and A.M. BROWN, Cir. Res. 47(6), 821 (1980). J.H. COOTE and Y. SATO, Cir. Res. 40, 571 (1973). C.T. HUANG, R. CARDONA and A.M. MICHELAKIS, Am. J. Physiol. 234, E25 (1978). H.D. BATTARBEE, L.E. SELF and G.E. FARRAR, Proc. Sot. Exptl. Biol. Med. l67, 182 (1981). G.L. WRIGHT, Can. J. Physiol. Pharmacol. 59, 1111 (1981). D.S. BLOOM, M.G. STEIN and C. ROSENDORFF, Cardiovas. Res. l0, 268 (1976). J.P. McMURTRY, G.L. WRIGHT and B.C. WEXLER, Science 211, 1173 (1981). G.L. WRIGHT and C. BOOKOUT, Can. J. Physiol. 60 622 (1982). A. GROLLMAN and V.S.R. KRIHNAMURTY, Am. J. Phziol. 221, 1499 (1971). L.T. SKEGGS, J.R. KAHN, M. LEVINE, F.E. DORER and K.E. LENTZ, Cir. Res. 40, 143 (1977). Y. HIRATA, L. TOBIAN, G. SIMON and J. IWAI, Hypertension 6, 709 (1984). J. YAMAMOTO, J. KIRA, M. MATSUMAGA, K. OGINO and C. KAWAI, Jpn. Cir. J. 3, 21 (1976). G.L. WRIGHT and W.D. McCUMBEE, Life Sci. 34, 1521 (1984). W.D. McCUMBEE and G.L. WRIGHT, Can. J. Physiol. Pharmacol. 63, 1321 (1985). W.D. McCUMBEE, P.J. JOHNSON, P.J. KASVINSKY and G.L. WRIGHT, Can. J. Physiol. Pharmacol. 65, 1991 (1987). G.L. WRIGHT, W.D. McCUMBEE, T. BROOME and S. EATON, J. Hypertension m, S145 (1986). G.L. WRIGHT and W.D. McCUMBEE (unpublished).
AN ENDOGENOUS PEPTIDE THAT INDUCES LONG-TERM BLOOD PRESSURE ELEVATION Gary L. Wright, Stephen Fish, Peter Johnson* and William D. McCumbee Departments of Physiology and Anatomy Barshall WrsiversitySchool of Medicine ttwltinqtan, WV 25704-2901 and *Department of Chemistry Ohio University Athens, DH 45701-2979 (Received in final form May 10, 1988) Summary A peptide was recently isolated from the blood of spontaneously hypertensive (SH) rats that stimulated an increase of calcium uptake by vascular tissue in vitro. In the present study normotensive rats were given nanomolar amounts of this peptide by intravenous or picomolar amounts by intracerebral injection and the effect on blood pressure was recorded. Injection of the peptide into the circulation had no significant effect on the elevation of blood pressure. By comparison, the injection of the compound into the third ventricle of the brain resulted in the elevation of blood> pressure to hypertensive levels. The blood pressure response was characterized by a prolonged period of onset with maximal elevation observed several days after the beginning of treatment. Subsequently, the increase in blood pressure was well maintained with significant elevation noted days following the cessation of treatment. The spontaneously hypertensive rat is a widely studied model of genetically programmed hypertension that provides close facsimiles of essential hypertension in the human. Hypertension in these rats is believed to be due primarily to peripheral vasoconstriction that is linked in some fashion to altered vessel smooth muscle contractility (1). The mechanisms of change in smooth muscle contractility is not certain, but there is some evidence suggesting that abnormal cellular handling of calcium is involved. Contractile dependency and sensitivity to extracellular calcium is increased in blood vessels of SH rats, a phenomenon possibly related to increased calcium permeability of the plasmalennna and reduced microsomal uptake of There is also a large body of calcium in SH rat smooth muscle (2-6). evidence that implicates both the central and peripheral nervous system in the development of hypertension. In the SH rat, the neurogenic component of hypertension may be the result of increased peripheral sympathetic neural activity (7-9). Several lines of evidence suggest that hypothalmic areas of the central nervous system are responsible for this activity which may be aggravated by impaired baroreflexes in the animal (10-12). The changes in vessel contractility and smooth muscle handling of calcium have been linked to the existence of a humoral substance in the SH and other models of hypertension. A number of laboratories have demonstrated 0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
112
Blood Pressure and an Endogenous Peptide
Vol. 43, No. 2, 1988
the presence of a plasma factor that sensitizes the vasculature to pressor agents (13-16), while other groups have reported hypertensive effects of Recently, an renal extracts and serum from hypertension models (17-22). extract of erythrocytes from SH rats was prepared that had a stimulatory effect on calcium uptake by vascular tissue _in vitro and a hypertensive effect when injected into normotensive rats (23,24). The compound having a stimulatory effect on calcium uptake has been isolated and tentatively identified as a small, acidic, calcium-dependent peptide with an amino acid composition of asp/asn (1.41), ser (1.02), glu/gln (1.00) and gly (2.00) (25). Based on the assumption that the peptide contained a single glutamic or glutamine residue, it was calculated that only nanomolar concentrations of materials were required to induce significant stimulation of calcium uptake. Methods In the present study we injected this peptide into 6 week old, male, Wistar Kyoto normotensive rats and observed its effect on blood pressure. The peptide was isolated from the erythrocytes of SH rats as previously The step necessary to completely eliminate phosphate described (25). contamination was deleted to ensure reasonable yields of the peptide. A portion of the sample was analyzed for glutamic/glutamine and phosphate content (25). Based on the assumption that the peptide contains one glu/gln residue, approximately 25 nmoles of the compound in 0.4 mL of distilled water (pH 7.0) was injected by tail vein. A second group of rats received 0.1 nmoles of peptide in 0.01 mL of 0.9% NaCl via a cannula to the third The cannula was implanted stereotaxically and ventricle of the brain. affixed to the skull with dental acrylic and screws. The cannula was sealed off between injections with a threaded cap. Controls received similar volumes of sodium phosphate buffer (pH 7.4). After blood pressure experiments were completed, the rats were given an overdose of nembutal and perfused through the heart with normal saline (150 mL) and 10% buffered formalin (300 mL). With the ventricular cannula left in place, the rostra1 calvaria and a portion of the dental acrylic cannula mount were dissected away. The cerebrum just rostra1 to the cannula was removed, along a frontal plane, so that the position of the cannula tip in the third ventricle could be verified. The brain was then completely removed and inspected for gross abnormalities. Three brains (one control and two experimental animals) were sectioned at 50 urn on a freezing microtome and prepared for microscopic analysis with the thionin stain. Blood pressure and the heart rate of each rat was determined without use of anesthetic using the Marco Programmed Electrosphygmomanometer with tail cuff and pneumatic transducer. The blood pressure was recorded at two day intervals for eight days prior to injections and the last three values were averaged as a preinjection control. Analysis of variance and Student's ttest were used to determine significant differences in the data. Results The mean blood pressure of normotensive rats receiving the peptide by intravenous injection was not significantly different from phosphate control values. In contrast those animals receiving single or multiple intracerebral injections of the peptide exhibited hypertensive elevation of blood pressure (Figs. 1 and 2). The rats given a single injection of peptide showed an average maximal elevation of blood pressure of about 25 torr, achieved at 3
Vol. 43, No. 2, 1988
Blood Pressure and an Endogenous Peptide
113
Fig. 1 Systolic blood pressure of Wistar Kyoto rats that were given a single injection of peptide (dashed line, open circle) or phosphate buffer (solid circle). Each of the peptide-treated group (n=6) received approximately 0.1 nanomole of the peptide in 0.01 mL of 0.9% NaCl, injected into the third ventricle of the brain. Controls (n=5) received a similar volume of phosphate buffer. The hatched area shows the mean + SEM systolic blood pressure of control rats prior to the injection of material. An asterisk or inverted cross indicates that values were significantly different from the preinjection value or phosphate injected control, respectively, p
114
Vol. 43, No. 2, 1988
Blood Pressure and an Endogenous Peptide
Gross and microscopic examination of the brains revealed no abnormalities except for minor widening of the ventricles near the cannulae. Discussion These findings suggest that the peptide isolated form the erythrocytes of SH rats, in addition to its potent stimulatory effect on calcium uptake in tissue may cause the elevation of blood pressure to hypertensive levels without a change in cardiac rate when injected into the brains of normotensive rats. The pressor response elicited appears unique in terms of the long
170-
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A
A-t,,
[
13o- a4e
I I 01
I 3
I 5
I
7
9
I
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I
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hY Fig. 2 Systolic blood pressure in Wistar Kyoto rats that were given daily intracerebral injection of peptide (dashed line) or phosphate Following the determination of preinjection buffer (solid line). control values, Group b rats (n=9) received injections of peptide for a period of 1 days after which phosphate buffer was injected. In a parallel experiment, Group fi rats (n=6) received phosphate buffer injections for 7 days followed by peptide for the remainder Horizontal bars indicate the mean f of the experimental period. As asterisk indicates a SEM of each group prior to injection. significant difference from the preinjection value, p
Vol. 43, No. 2, 1988
Blood Pressure and an Endogenous Peptide
115
The interval required for development and the prolonged recovery period. mechanism of the peptide-induced increase in blood pressure may be linked with the compound's influence on tissue metabolism of calcium. However, the discrepancy between the time required for evidence of pressor activity (days) and stimulation of calcium uptake (minutes) renders the relationship unclear. An alternative explanation is that the elevation in blood pressure noted was an artifact of localized damage and CNS lesions leading to hypertension. However, an examination of brains revealed no apparent differences between control and peptide-treated groups. The recovery of peptide injected animals (Fig. 1) and lack of symptoms other than elevation of blood pressure also indicates this explanation is unlikely. The finding that the purified peptide had little effect when given by intravenous injection was surprising in light of previous observations of severe hypertension induced by semipurified preparations given via this route (23,24). One explanation of the apparent loss of activity is that the amount of material injected was below a threshold level for biological action due to peptide degradation, sequestration and/or excretion in the peripheral circulation. Alternately, a carrier molecule or peptide-cofactor may have been lost in the final steps of purification. In any case the administration of the peptide by intracerebral cannula was clearly more effective in inducing blood pressure elevation than intravenous injection of the compound. While the results indicate that the peptide can cause the elevation of blood pressure, the significance of the compound to hypertension in the SH rat is less clear. The peptide has been shown to be present in normotensive animals. However, the yield of the compound from erythrocytes of SH rats is consistently 2 to 5 fold greater than from normotensive rats (23). Dietary manipulation which attenuates blood pressure in SH rats has been found to cause a significant reduction in blood levels of the peptide (26). Normotensive rats subjected to the same dietary regimen, on the other hand, did not show an effect on blood pressure nor in the levels of calcium stimulator activity. Additionally, the blood levels of calcium stimulator activity in 5 week old SH rats, prior to a significant rise in blopd pressure, was not different from that noted in similarly aged normotensive rats (27). Hence, the increase in concentration of the peptide that occurs following maturation and the rise of blood pressure in SH rats, is absent in normotensive rats that do not undergo hypertensive elevation of blood pressure after maturation. Taken as a whole, these observations suggest that the peptide may play a role in the development of high blood pressure in the SH rat. References
:: 3. 4.
ii: fi 9.
D.F. BOHR, Fed. Proc. 33, 127 (1974). O.L. PEDERSEN, Arch. Int. Pharmacodyn. de Therap. 239, 208 (1979). R.C. BHALLA, R.C. WEBB, D. SINGH, T. ASHLEY and T. BROCK, Molt. Pharmacol. I4, 468 (1978). K. AOKI, Y. YAMASHITA, A. SUZUKI, K. TAKIKAWA and K. HOTTA, Clin. Exptl. Pharmacol. Physiol. 3, 27 (1976). S. SHIBATA, M. KUCHII, and T. TANIGUCHI, Blood Vessels l2, 279 (1975). C.Y. KWAN, Can. J. Physiol. Pharmacol. 63, 366 (1985). W.V. JUDY, A.M. WATANABE and D.P. HENRY, Cir. Res. 38 2 , 21 (1976). A. NAGAOKA and W. LOVENBERG, Life Sci. l9, 29 (1977F . M.F. ROIZEN, V. WEISE and H. GROBECKER, Life Sci. 17(2), 283 (1975).
116
10.
:::
13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Blood Pressure and an Endogenous Peptide
Vol. 43, No. 2, 1988
J. CIRJELLO, R.L. KLINE, T.X. ZANG and M.M. CAVERSON, Brain Res. 310, 355 (1984). M.C. ANDRESEN, S. KURAOKA and A.M. BROWN, Cir. Res. 47(6), 821 (1980). J.H. COOTE and Y. SATO, Cir. Res. 40, 571 (1973). C.T. HUANG, R. CARDONA and A.M. MICHELAKIS, Am. J. Physiol. 234, E25 (1978). H.D. BATTARBEE, L.E. SELF and G.E. FARRAR, Proc. Sot. Exptl. Biol. Med. l67, 182 (1981). G.L. WRIGHT, Can. J. Physiol. Pharmacol. 59, 1111 (1981). D.S. BLOOM, M.G. STEIN and C. ROSENDORFF, Cardiovas. Res. l0, 268 (1976). J.P. McMURTRY, G.L. WRIGHT and B.C. WEXLER, Science 211, 1173 (1981). G.L. WRIGHT and C. BOOKOUT, Can. J. Physiol. 60 622 (1982). A. GROLLMAN and V.S.R. KRIHNAMURTY, Am. J. Phziol. 221, 1499 (1971). L.T. SKEGGS, J.R. KAHN, M. LEVINE, F.E. DORER and K.E. LENTZ, Cir. Res. 40, 143 (1977). Y. HIRATA, L. TOBIAN, G. SIMON and J. IWAI, Hypertension 6, 709 (1984). J. YAMAMOTO, J. KIRA, M. MATSUMAGA, K. OGINO and C. KAWAI, Jpn. Cir. J. 3, 21 (1976). G.L. WRIGHT and W.D. McCUMBEE, Life Sci. 34, 1521 (1984). W.D. McCUMBEE and G.L. WRIGHT, Can. J. Physiol. Pharmacol. 63, 1321 (1985). W.D. McCUMBEE, P.J. JOHNSON, P.J. KASVINSKY and G.L. WRIGHT, Can. J. Physiol. Pharmacol. 65, 1991 (1987). G.L. WRIGHT, W.D. McCUMBEE, T. BROOME and S. EATON, J. Hypertension m, S145 (1986). G.L. WRIGHT and W.D. McCUMBEE (unpublished).