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Structure-cardiovascular activity relationships of parathyroid hormone
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
86, 506-510 (1992)
Structure-Cardiovascular Activity Relationships Parathyroid Hormone
of
K. W. CHIU, Y. C. LEE AND P. K. T. PANG* Department of Biology, The Chinese University of Hong Kong, Hong Kong; and *Department University of Alberta, Canada
of
Physiology,
Accepted September 17, 1991 The effects of parathyroid hormone (PTH) analogues on isolated rat tail arteries and frog arteries and heart were examined. Synthetic bovine PTH fragment 1 to 34 [bPTH-(l-34)], porcine PTH-(l-34), [Ala’] pPTH-(l-34), and [Nle’] pPTH-(1-34) were tested for vasorelaxant activity on helical strips of the rat tail artery and the frog iliac and femoral artery, preconstricted with arginine vasopressin or arginine vasotocin in the rat and the frog, respectively. For cardiac activity, PTH analogues were administered to isolated frog atria, and atria1 rate and atrial tension (AT) were measured. The N-terminal amino acid alanine appears to be pivotal for vasorelaxant activity of PTH molecules on the rat tail artery strips. Data from the frog preparations supported the finding in the rat, although the iliac and femoral arterial preparations differed in their responses. The N-terminal serine in pPTH(l-34), compared with the other PTH analogues, appeared to be responsible for different cardiac responses, being negatively chronotropic and without effect on AT. o 1992Academic Press, Inc.
Parathyroid hormone (PTH) has welldocumented hypotensive and hypercalcaemic effects in mammals (see Mok et al., 1989). These two activities seem to lie in different parts of the PTH molecule, the structure of which is known for a number of mammalian species including human; synthetic fragments and analogues of the molecule are available. Bovine PTH-( l-34) possesses the full hypotensive and hypercalcemic activity of the whole molecule bPTH-(l-84), and attempts have been made to establish the structure-activity relationship. The hypotensive action seems to reside in the bPTH-(24-28) sequence (Hong et al., 1986). However, bPTH-(3-34) and bPTH-(7-34) are biologically inactive, and have been used as antagonists. Also [Nle**“, Tyr34] bPTH-(3-34) has no effect in doses up to 500 yg/kg on blood pressure (Ellison and McCarron, 1984). It has been argued therefore that the N-terminus of the molecule is important, and doubts have
been raised over the importance of the pentapeptide bPTH-(24-28). The present study provides further data on the structureactivity relationships of PTH in the rat and frog. MATERIALS Rat Vasorelaxant effects. Male Sprague-Dawley rats (300-350 g) were killed by decapitation. This was done in accord with codes and in a humane manner. The ventral caudal artery was rapidly isolated and placed in aerated Krebs-Henseleit solution (KHS in n-&f: NaCl, 115; KCl, 5; CaCl,, 2.1; NaHCO,, 25; NaH,PO,, 1.2; MgSO, 7H20, 1.2; glucose, 11 pH 7.4,340 mOsm). Segments of about 2 cm of the artery were cut into helical strips and suspended inside an organ bath containing 10 ml KHS aerated with 95% 0,:5% CO, and maintained at 37”. The two ends of the helical strip were tied with silk threads and connected to the bottom of the bath with a force displacement transducer above. Isometric contractions were recorded on a polygraph. Before exposure to stimulants, the helical strip was equilibrated for 60 min at a resting tension of 0.7g. The helical strip was tested with 60 506
0016~6480/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
PARATHYROID
HORMONE
mM KCl. An acceptable strip should increase in tension by 0.5 to l.Og,, and KCl-induced contraction should be stable after three tests with KCl. After each test, the helical strip was washed several times with fresh KHS and given a lo-min equilibration. KC1 (60 mM), norepinephrine (NE, 3 x 10-M), and arginine vasopressin (AVP, 2.11 x IO-‘M, 3 ngknl) were used as preconstrictors and were added directly to the bath. Preliminary data indicated that AVP-preconstricted rat tail artery strips responded best to pPTH-(1-34). This nonapeptide was therefore chosen as the preconstrictor in the present study on the vasorelaxant effects of the PTH analogues in the rat caudal arterial strips. To test the effect of the PTH analogues, the helical strips were first preconstricted with AVP (3 rig/ml). The response consisted of a rapid rise in tension reaching a maximum value, followed by either an immediate steady decline or a period of time at the maxima prior to declining. The time course of changes in tension over 15 min was recorded to serve as control. The strips were washed several times with KHS and equilibrated for 60 min before being challenged with AVP for the second time. When a steady tension was achieved, the lowest dose of a PTH analogue was added. As soon as a drop in induced tension became steady, another (the next higher) dose of the PTH analogue was added cumulatively. This procedure was repeated. Four doses were tested (30, 100, 300, and 1000 @ml [3.15 X 10e9, 1.05 x 10e8, 3.15 x 10M8, and 1.05 X lo-‘M, respectively]). The cumulative dose responses were obtained.
STRUCTURE
AND
ACTIVITY
7
brate for 30 min prior to being tested for use, as in the rat caudai arterial strips, with 60 mM KCI. A minim& increase of about 0.31: in tension was required from the strip in order to be used further. To test the effects of the PTH analogues, the helical strips were first preconstricted with arginine vasotocin (AVT, 2.86 x lo-‘M, 30 rig/ml). The time course in tension changes within a IO-min interval was recorde as control. The tissues were then rinsed and allowed t recover for at least 40 min before more AVT IY~S added to constrict the strips. When a sustained force. or a steady but constant rate of decrease in force, had developed. a dose of PTH analogue was added to the bath and the changes in tension were recorded for IO min. Tissues were then rinsed and allowed to recover for at least 40 min before the procedures of preconstriction with AVT and the testing of another dose of the PTH analogue. The difference in the extent of the tension change, between the control (xg) and that measured in the presence of the PTH analogue (yg), i.e., the effect induced by the analogue ](x - y)g], was compiled and the ratio of (x - y)/.r was expressed as percentage change in tension. The following PTH analogues wer the present study: bovine PTH-(l-34), po 4 I34), [Ala’] pPTH-(1-34). and [Nle’] 34). These analogues were synthesized in the ~~oc~~rnist~y Department, University of Alberta, Canada. All data were expressed as means i SE cal comparisons were made by unpaired Student’s 1 test (Zar, 1974). A P value of <0.05 was regarded as statistically significant.
Frog Cardiac effecrs. Tiger frogs, Ram tigrina, of both sexes were obtained in October. Animals were doublepithed and hearts were rapidly excised and transferred to Petri dishes containing aerated frog Ringer’s solution (FRS in mM: NaCl, 80; KCl, 2.5; CaCl,, 1.8; NaHCO,, 24; NaH,PO,, 0.12; glucose, 1.1). Paired atria were isolated and suspended in an organ bath with 10 ml FRS aerated with 95% 0,:5% CO,. The bath was held at room temperature (23”). Atria were preloaded at a resting tension of lg, washed, and equilibrated for at least 30 min. When steady atria1 rates (basal, AR) and atrial tensions (basal, AT) were attamed, a PTH analogue was added to the bath in cumulative doses. These atria were tested for one PTH analogue only. A dose-response curve was constructed. Vusorelaxunt effects. The iliac and femoral arteries were rapidly removed from the double-pithed frogs obtained in October-January. These were cleaned in the petri dishes with aerated FRS. These arteries, about 2 cm in length, were cut into helical strips and suspended in an organ bath with aerated FRS at a resting tension of 0.7Sg. The tissues were allowed to equili-
Rat The percentage decreases in tension of the AVP-preconstricted caudal arterial strips are shown in Fig. 1. Of the alogues tested, bPTH-(1-34) was active, inducing a decrease of abou 300 nglml. The dose-response G [Ala’] pPTH-( l--34), pPT [Nle*] pPTH-( C-34) were to for bPTH-( l-34). Frog The percentage decreases in the AVT-preconstricted iliac a arterial strips are shown in F respectively bPTH-(l-34) and [ (l-34) produced a decrease of about 25% in
508
CHIU,
0 -0 . -.
CONCENTRATION
( Xl 0-‘ON
en4 (n=Z6, pm. (F31,
&.A .-.
LEE,
[/do~]-pPIH cn= 5) [NieB]-m b=jlJ
FIG. 1. Vasorelaxant effects of PTH analogues on the rat tail artery. Vertical bars represent SE. n, number of preparations.
tension at 100 and 10 rig/ml, respectively, in the iliac and femoral arterial strips. pPTH(l-34) had no effect on the iliac artery, but relaxed the preconstricted femoral artery. In the latter preparations, both bPTH-(l34) and [Ala’] pPTH-(1-34) induced an initial decrease in tension of about 15% at 1 rig/ml. The effect of [Nle’] pPTH-(1-34) was variable in both iliac and femoral arteries, and did not give dose-related responses. The chronotropic and inotropic effects of the PTH analogues on the isolated frog atria are shown in Figs. 4 and 5. pPTH-(1-34) significantly decreased the AR. The other PTH analogues had no effects. The reverse CONCENTRATION
AND
PANG
CONCENTRATION
)
FIG. 3. Vasorelaxant effects of PTH analogues on the frog fermoral artery. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with control.
was true on the AT responses; pPTH-( l-34) did not alter the AT, while the other PTH analogues significantly increased it. The maximal AT increases, ranging from 0.25 to 0.55g, were observed at 100 rig/ml. DISCUSSION
Rat
The importance of the N-terminus for the biological activity of the PTH molecule in the mammal has been reconfirmed. The present data indicate that this might be due to the presence of the terminal amino acid, alanine. Primary structural differences between bPTH-(1-34) and pPTH-(1-34) are few, namely Ala’ Phe7 Met’* in bPTH-(l34) and Ser’ Leu7,‘* in pPTH-(1-34). For
( X10-‘%
CONCENTRATION
2. Vasorelaxant effects of PTH analogues on the frog iliac artery. *, P < 0.05; **, P ==z0.01, compared with control. FIG.
( Xl 0-“M
( Xl O-I’M
)
4. Chronotropic effects of the PTH analogues on isolated frog atria. *, P < 0.05; **, P < 0.01, compared with basal AR. FIG.
PARATHYROID
CONCENTRATION
( X1 0-“M
HORMONE
)
FIG. 5. Inotropic effects of the PTH anaiogues on isolated frog atria. *, P < 0.05; **, P < 0.01, compared with basal AT.
the hypotensive activity, alanine is required and its methyl side chain seems important when bPTH-( l-34) interacts with its receptor. When it is replaced by, for example, Ser in pPTH-(l-34), the activity is greatly reduced. Thus when Set-’ of pPTH-( l-34) is replaced with Ala’ to become Ala’ pPTH(I-34), full activity of the molecule is restored. This also explains earlier findings that bPTH-(3-34) is not active because the analogue begins with Ser. Ser carries a side chain of an OH-CH, group and thus differs from Ala. By the same token, bPTH-(7-34) is not active, since Phe7 carries a phenolic ring. If the presence of a methyl group on the side chain of the N-terminal amino acid is indeed important, it is understandable why the active core, bPTH-(24-28) in the series of peptides tested exhibits significant activity. This may reflect the fact that the side chain of Leu’4 is comparable to that of nine, a methyl residue. It might be coned that the “‘normal” vasorelaxant activity of PTH is the result of at least the pentapeptide bPTH-(24-28) plus Ala’ of the molecule. Frog The importance of the N-terminal amino acid alanine in the vasorelaxant activity of is again shown. There is no difference in the response of frog blood vessels to
STRUCTURE
AND
ACTIVITY
5
bPTH-(1-34) and [Alar] pPT There is however a difference in the response of the AVT-preco strips to pPTH-(l-34) between the and the iliac arteries. In the latter (l-34) is ineffective while i vasoactive; this probably indicates ences in the nature or distributio receptors. It is of interest t artery is highly sensitive to which there is an initial drop of at 1 ngiml of bPTH-(1-34) or {Al (l-34), a finding earlier reported fects of bPTH-(l-34) on the frog b~~~~ vessels (Chiu et al., 1991). The vasorelaxant data in the rat and frog may not be appropriately compared since there are obvious differences iological/pharmacological res present study, this is further with the use of a mammalian the frog. The structure of frog known Molecular variations between ties and indeed within a species are c mon with this peptide. ~asoact~v~t~ o bPTII-(I-34), however, has been the frog (Furspan et al., 198 1984; Chiu et al., 1990) and in other vertebrates (Crass and Pang, 1980; Lhoste et wl., 1980; Tenner and ng, 1982; Temer et ai. p 1983; Sham and g, 1986; Sham et al., 1986).
Finally, the increases in the c~~t~act~~~ response of the atria1 preparations induced by PTH analogues contrast ant responses Except for p PTH analogues produce a positive inotropit response on isolated atria. is the most effective, and a do sponse is obtained. There is n
pPTH-( l-34) or other
510
CHIU,
LEE, AND PANG
notropic effect on isolated atria during the months of February, May, August, and December (Chiu et al., 1990), although an unexpected negative chronotropic response occurs in July, but not in June or August (Chiu et al., 1991). REFERENCES Chiu, K. W., Lee, Y. C., and Pang, P. K. T. (1990). The cardiac effects of parathyroid hormone in the tiger frog, Rana tigrina. Comp. Biochem. Physiol. c 95, 197-199. Chiu, K. W., Chung, T. Y., and Pang, P. K. T. (1991). Cardiovascular effects of bPTH-(l-34) and isoproterenol in the frog, Rana tigrina. Comp. B&hem. Physiol. lOOC, 547-552. Crass M. F. III, and Pang, P. K. T. (1980). Parathyroid hormone: A coronary vasodilator. Science 207, 1087-1089. Ellison, D., and McCarron, D. A. (1984). Structural prerequisites for the hypotensive action of parathyroid hormone. Am. J. Physiol. 246, F551-556. Furspan, P. B., Sham, J. S. K., Shew, R. L., Peng, G., and Pang, P. K. T. (1984). Cardiac actions of parathyroid hormones in some vertebrates. Gen. Comp. Endocrinol. 56, 246-251. Hong, B. S., Yang, M. C. M., Liang, J. N., and Pang, P. K. T. (1986). Correlation of structural changes in parathyroid hormone with its vascular action. Peptides 7, 1131.
Lhoste, F., Drueke, T., Lamo, S., and Boissier, J. R. (1980). Cardiac interaction between parathyroid hormone, B-adrenoceptor agents and verapamil in guinea pig in vitro. Clin. Exp. Pharmacol. Physiol. 7, 119-127. Mok, L. L. S., Nickels, A. G., Thompson, J. C., and Cooper, G. W. (1989). Parathyroid hormone as a smooth muscle relaxant. Endocr. Rev. 10, 420436. Sham, J. S. K., and Pang, P. K. T. (1986). Calciumdependent chronotropic action of bovine parathyroid hormone (l-34) in isolated atria of Japanese quail, Coturnix coturnix japonica. Gen. Comp. Endocrinol.
64, 250-253.
Sham, J. S. K., Kenny, A. D., and Pang, P. K. T. (1984). Cardiac actions and structural-activity relationship of parathyroid hormone on isolated frog atrium. Gen. Comp. Endocrinol. 55, 373-377. Sham, J. S. K., Wong, V. C. K., Chiu, K. W., and Pang, P. K. T. (1986). Comparative study on the cardiac actions of bovine parathyroid hormone (l-34). Gen. Comp. Endocrinol. 61, 148-152. Tenner, T. E. Jr., and Pang, P. K. T. (1982). Cardiac actions of parathyroid hormone. Proc. West. Pharmacol.
Sot.
25, 263-267.
Tenner, T. E. Jr., Ramanadham, S., Yang, M. C. M., and Pang, P. K. T. (1983). Chronotropic actions of bPTH-( l-34) in the right atrium of the rat. Can. J. Physiol. Pharmacol. 61, 1162-1167. Zar, J. H. (1974). “Biostatistical Analysis.” Prentice Hall, New York.
AND
COMPARATIVE
ENDOCRINOLOGY
86, 506-510 (1992)
Structure-Cardiovascular Activity Relationships Parathyroid Hormone
of
K. W. CHIU, Y. C. LEE AND P. K. T. PANG* Department of Biology, The Chinese University of Hong Kong, Hong Kong; and *Department University of Alberta, Canada
of
Physiology,
Accepted September 17, 1991 The effects of parathyroid hormone (PTH) analogues on isolated rat tail arteries and frog arteries and heart were examined. Synthetic bovine PTH fragment 1 to 34 [bPTH-(l-34)], porcine PTH-(l-34), [Ala’] pPTH-(l-34), and [Nle’] pPTH-(1-34) were tested for vasorelaxant activity on helical strips of the rat tail artery and the frog iliac and femoral artery, preconstricted with arginine vasopressin or arginine vasotocin in the rat and the frog, respectively. For cardiac activity, PTH analogues were administered to isolated frog atria, and atria1 rate and atrial tension (AT) were measured. The N-terminal amino acid alanine appears to be pivotal for vasorelaxant activity of PTH molecules on the rat tail artery strips. Data from the frog preparations supported the finding in the rat, although the iliac and femoral arterial preparations differed in their responses. The N-terminal serine in pPTH(l-34), compared with the other PTH analogues, appeared to be responsible for different cardiac responses, being negatively chronotropic and without effect on AT. o 1992Academic Press, Inc.
Parathyroid hormone (PTH) has welldocumented hypotensive and hypercalcaemic effects in mammals (see Mok et al., 1989). These two activities seem to lie in different parts of the PTH molecule, the structure of which is known for a number of mammalian species including human; synthetic fragments and analogues of the molecule are available. Bovine PTH-( l-34) possesses the full hypotensive and hypercalcemic activity of the whole molecule bPTH-(l-84), and attempts have been made to establish the structure-activity relationship. The hypotensive action seems to reside in the bPTH-(24-28) sequence (Hong et al., 1986). However, bPTH-(3-34) and bPTH-(7-34) are biologically inactive, and have been used as antagonists. Also [Nle**“, Tyr34] bPTH-(3-34) has no effect in doses up to 500 yg/kg on blood pressure (Ellison and McCarron, 1984). It has been argued therefore that the N-terminus of the molecule is important, and doubts have
been raised over the importance of the pentapeptide bPTH-(24-28). The present study provides further data on the structureactivity relationships of PTH in the rat and frog. MATERIALS Rat Vasorelaxant effects. Male Sprague-Dawley rats (300-350 g) were killed by decapitation. This was done in accord with codes and in a humane manner. The ventral caudal artery was rapidly isolated and placed in aerated Krebs-Henseleit solution (KHS in n-&f: NaCl, 115; KCl, 5; CaCl,, 2.1; NaHCO,, 25; NaH,PO,, 1.2; MgSO, 7H20, 1.2; glucose, 11 pH 7.4,340 mOsm). Segments of about 2 cm of the artery were cut into helical strips and suspended inside an organ bath containing 10 ml KHS aerated with 95% 0,:5% CO, and maintained at 37”. The two ends of the helical strip were tied with silk threads and connected to the bottom of the bath with a force displacement transducer above. Isometric contractions were recorded on a polygraph. Before exposure to stimulants, the helical strip was equilibrated for 60 min at a resting tension of 0.7g. The helical strip was tested with 60 506
0016~6480/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
PARATHYROID
HORMONE
mM KCl. An acceptable strip should increase in tension by 0.5 to l.Og,, and KCl-induced contraction should be stable after three tests with KCl. After each test, the helical strip was washed several times with fresh KHS and given a lo-min equilibration. KC1 (60 mM), norepinephrine (NE, 3 x 10-M), and arginine vasopressin (AVP, 2.11 x IO-‘M, 3 ngknl) were used as preconstrictors and were added directly to the bath. Preliminary data indicated that AVP-preconstricted rat tail artery strips responded best to pPTH-(1-34). This nonapeptide was therefore chosen as the preconstrictor in the present study on the vasorelaxant effects of the PTH analogues in the rat caudal arterial strips. To test the effect of the PTH analogues, the helical strips were first preconstricted with AVP (3 rig/ml). The response consisted of a rapid rise in tension reaching a maximum value, followed by either an immediate steady decline or a period of time at the maxima prior to declining. The time course of changes in tension over 15 min was recorded to serve as control. The strips were washed several times with KHS and equilibrated for 60 min before being challenged with AVP for the second time. When a steady tension was achieved, the lowest dose of a PTH analogue was added. As soon as a drop in induced tension became steady, another (the next higher) dose of the PTH analogue was added cumulatively. This procedure was repeated. Four doses were tested (30, 100, 300, and 1000 @ml [3.15 X 10e9, 1.05 x 10e8, 3.15 x 10M8, and 1.05 X lo-‘M, respectively]). The cumulative dose responses were obtained.
STRUCTURE
AND
ACTIVITY
7
brate for 30 min prior to being tested for use, as in the rat caudai arterial strips, with 60 mM KCI. A minim& increase of about 0.31: in tension was required from the strip in order to be used further. To test the effects of the PTH analogues, the helical strips were first preconstricted with arginine vasotocin (AVT, 2.86 x lo-‘M, 30 rig/ml). The time course in tension changes within a IO-min interval was recorde as control. The tissues were then rinsed and allowed t recover for at least 40 min before more AVT IY~S added to constrict the strips. When a sustained force. or a steady but constant rate of decrease in force, had developed. a dose of PTH analogue was added to the bath and the changes in tension were recorded for IO min. Tissues were then rinsed and allowed to recover for at least 40 min before the procedures of preconstriction with AVT and the testing of another dose of the PTH analogue. The difference in the extent of the tension change, between the control (xg) and that measured in the presence of the PTH analogue (yg), i.e., the effect induced by the analogue ](x - y)g], was compiled and the ratio of (x - y)/.r was expressed as percentage change in tension. The following PTH analogues wer the present study: bovine PTH-(l-34), po 4 I34), [Ala’] pPTH-(1-34). and [Nle’] 34). These analogues were synthesized in the ~~oc~~rnist~y Department, University of Alberta, Canada. All data were expressed as means i SE cal comparisons were made by unpaired Student’s 1 test (Zar, 1974). A P value of <0.05 was regarded as statistically significant.
Frog Cardiac effecrs. Tiger frogs, Ram tigrina, of both sexes were obtained in October. Animals were doublepithed and hearts were rapidly excised and transferred to Petri dishes containing aerated frog Ringer’s solution (FRS in mM: NaCl, 80; KCl, 2.5; CaCl,, 1.8; NaHCO,, 24; NaH,PO,, 0.12; glucose, 1.1). Paired atria were isolated and suspended in an organ bath with 10 ml FRS aerated with 95% 0,:5% CO,. The bath was held at room temperature (23”). Atria were preloaded at a resting tension of lg, washed, and equilibrated for at least 30 min. When steady atria1 rates (basal, AR) and atrial tensions (basal, AT) were attamed, a PTH analogue was added to the bath in cumulative doses. These atria were tested for one PTH analogue only. A dose-response curve was constructed. Vusorelaxunt effects. The iliac and femoral arteries were rapidly removed from the double-pithed frogs obtained in October-January. These were cleaned in the petri dishes with aerated FRS. These arteries, about 2 cm in length, were cut into helical strips and suspended in an organ bath with aerated FRS at a resting tension of 0.7Sg. The tissues were allowed to equili-
Rat The percentage decreases in tension of the AVP-preconstricted caudal arterial strips are shown in Fig. 1. Of the alogues tested, bPTH-(1-34) was active, inducing a decrease of abou 300 nglml. The dose-response G [Ala’] pPTH-( l--34), pPT [Nle*] pPTH-( C-34) were to for bPTH-( l-34). Frog The percentage decreases in the AVT-preconstricted iliac a arterial strips are shown in F respectively bPTH-(l-34) and [ (l-34) produced a decrease of about 25% in
508
CHIU,
0 -0 . -.
CONCENTRATION
( Xl 0-‘ON
en4 (n=Z6, pm. (F31,
&.A .-.
LEE,
[/do~]-pPIH cn= 5) [NieB]-m b=jlJ
FIG. 1. Vasorelaxant effects of PTH analogues on the rat tail artery. Vertical bars represent SE. n, number of preparations.
tension at 100 and 10 rig/ml, respectively, in the iliac and femoral arterial strips. pPTH(l-34) had no effect on the iliac artery, but relaxed the preconstricted femoral artery. In the latter preparations, both bPTH-(l34) and [Ala’] pPTH-(1-34) induced an initial decrease in tension of about 15% at 1 rig/ml. The effect of [Nle’] pPTH-(1-34) was variable in both iliac and femoral arteries, and did not give dose-related responses. The chronotropic and inotropic effects of the PTH analogues on the isolated frog atria are shown in Figs. 4 and 5. pPTH-(1-34) significantly decreased the AR. The other PTH analogues had no effects. The reverse CONCENTRATION
AND
PANG
CONCENTRATION
)
FIG. 3. Vasorelaxant effects of PTH analogues on the frog fermoral artery. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with control.
was true on the AT responses; pPTH-( l-34) did not alter the AT, while the other PTH analogues significantly increased it. The maximal AT increases, ranging from 0.25 to 0.55g, were observed at 100 rig/ml. DISCUSSION
Rat
The importance of the N-terminus for the biological activity of the PTH molecule in the mammal has been reconfirmed. The present data indicate that this might be due to the presence of the terminal amino acid, alanine. Primary structural differences between bPTH-(1-34) and pPTH-(1-34) are few, namely Ala’ Phe7 Met’* in bPTH-(l34) and Ser’ Leu7,‘* in pPTH-(1-34). For
( X10-‘%
CONCENTRATION
2. Vasorelaxant effects of PTH analogues on the frog iliac artery. *, P < 0.05; **, P ==z0.01, compared with control. FIG.
( Xl 0-“M
( Xl O-I’M
)
4. Chronotropic effects of the PTH analogues on isolated frog atria. *, P < 0.05; **, P < 0.01, compared with basal AR. FIG.
PARATHYROID
CONCENTRATION
( X1 0-“M
HORMONE
)
FIG. 5. Inotropic effects of the PTH anaiogues on isolated frog atria. *, P < 0.05; **, P < 0.01, compared with basal AT.
the hypotensive activity, alanine is required and its methyl side chain seems important when bPTH-( l-34) interacts with its receptor. When it is replaced by, for example, Ser in pPTH-(l-34), the activity is greatly reduced. Thus when Set-’ of pPTH-( l-34) is replaced with Ala’ to become Ala’ pPTH(I-34), full activity of the molecule is restored. This also explains earlier findings that bPTH-(3-34) is not active because the analogue begins with Ser. Ser carries a side chain of an OH-CH, group and thus differs from Ala. By the same token, bPTH-(7-34) is not active, since Phe7 carries a phenolic ring. If the presence of a methyl group on the side chain of the N-terminal amino acid is indeed important, it is understandable why the active core, bPTH-(24-28) in the series of peptides tested exhibits significant activity. This may reflect the fact that the side chain of Leu’4 is comparable to that of nine, a methyl residue. It might be coned that the “‘normal” vasorelaxant activity of PTH is the result of at least the pentapeptide bPTH-(24-28) plus Ala’ of the molecule. Frog The importance of the N-terminal amino acid alanine in the vasorelaxant activity of is again shown. There is no difference in the response of frog blood vessels to
STRUCTURE
AND
ACTIVITY
5
bPTH-(1-34) and [Alar] pPT There is however a difference in the response of the AVT-preco strips to pPTH-(l-34) between the and the iliac arteries. In the latter (l-34) is ineffective while i vasoactive; this probably indicates ences in the nature or distributio receptors. It is of interest t artery is highly sensitive to which there is an initial drop of at 1 ngiml of bPTH-(1-34) or {Al (l-34), a finding earlier reported fects of bPTH-(l-34) on the frog b~~~~ vessels (Chiu et al., 1991). The vasorelaxant data in the rat and frog may not be appropriately compared since there are obvious differences iological/pharmacological res present study, this is further with the use of a mammalian the frog. The structure of frog known Molecular variations between ties and indeed within a species are c mon with this peptide. ~asoact~v~t~ o bPTII-(I-34), however, has been the frog (Furspan et al., 198 1984; Chiu et al., 1990) and in other vertebrates (Crass and Pang, 1980; Lhoste et wl., 1980; Tenner and ng, 1982; Temer et ai. p 1983; Sham and g, 1986; Sham et al., 1986).
Finally, the increases in the c~~t~act~~~ response of the atria1 preparations induced by PTH analogues contrast ant responses Except for p PTH analogues produce a positive inotropit response on isolated atria. is the most effective, and a do sponse is obtained. There is n
pPTH-( l-34) or other
510
CHIU,
LEE, AND PANG
notropic effect on isolated atria during the months of February, May, August, and December (Chiu et al., 1990), although an unexpected negative chronotropic response occurs in July, but not in June or August (Chiu et al., 1991). REFERENCES Chiu, K. W., Lee, Y. C., and Pang, P. K. T. (1990). The cardiac effects of parathyroid hormone in the tiger frog, Rana tigrina. Comp. Biochem. Physiol. c 95, 197-199. Chiu, K. W., Chung, T. Y., and Pang, P. K. T. (1991). Cardiovascular effects of bPTH-(l-34) and isoproterenol in the frog, Rana tigrina. Comp. B&hem. Physiol. lOOC, 547-552. Crass M. F. III, and Pang, P. K. T. (1980). Parathyroid hormone: A coronary vasodilator. Science 207, 1087-1089. Ellison, D., and McCarron, D. A. (1984). Structural prerequisites for the hypotensive action of parathyroid hormone. Am. J. Physiol. 246, F551-556. Furspan, P. B., Sham, J. S. K., Shew, R. L., Peng, G., and Pang, P. K. T. (1984). Cardiac actions of parathyroid hormones in some vertebrates. Gen. Comp. Endocrinol. 56, 246-251. Hong, B. S., Yang, M. C. M., Liang, J. N., and Pang, P. K. T. (1986). Correlation of structural changes in parathyroid hormone with its vascular action. Peptides 7, 1131.
Lhoste, F., Drueke, T., Lamo, S., and Boissier, J. R. (1980). Cardiac interaction between parathyroid hormone, B-adrenoceptor agents and verapamil in guinea pig in vitro. Clin. Exp. Pharmacol. Physiol. 7, 119-127. Mok, L. L. S., Nickels, A. G., Thompson, J. C., and Cooper, G. W. (1989). Parathyroid hormone as a smooth muscle relaxant. Endocr. Rev. 10, 420436. Sham, J. S. K., and Pang, P. K. T. (1986). Calciumdependent chronotropic action of bovine parathyroid hormone (l-34) in isolated atria of Japanese quail, Coturnix coturnix japonica. Gen. Comp. Endocrinol.
64, 250-253.
Sham, J. S. K., Kenny, A. D., and Pang, P. K. T. (1984). Cardiac actions and structural-activity relationship of parathyroid hormone on isolated frog atrium. Gen. Comp. Endocrinol. 55, 373-377. Sham, J. S. K., Wong, V. C. K., Chiu, K. W., and Pang, P. K. T. (1986). Comparative study on the cardiac actions of bovine parathyroid hormone (l-34). Gen. Comp. Endocrinol. 61, 148-152. Tenner, T. E. Jr., and Pang, P. K. T. (1982). Cardiac actions of parathyroid hormone. Proc. West. Pharmacol.
Sot.
25, 263-267.
Tenner, T. E. Jr., Ramanadham, S., Yang, M. C. M., and Pang, P. K. T. (1983). Chronotropic actions of bPTH-( l-34) in the right atrium of the rat. Can. J. Physiol. Pharmacol. 61, 1162-1167. Zar, J. H. (1974). “Biostatistical Analysis.” Prentice Hall, New York.