AMEKKAN JOURNAL OF PHYSIOLOGY Vol. 228, No. 5, May 1975. Printed in U.S.A.
Magnesium-neurohypophyseal in contraction
hormone
of vascular
smooth
interactions
muscle
BURTON M. ALTURA Departments of Anesthesiology and Physiology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, and Department of Physiology, State University of .A New York Downstate Medical Center, Brooklyn, New York 11203
ALTURA, BURTON R/I. Mag~sium-neurohypuphyseal hormone interactions in contraction of vascular smooth muscle. Am. J. Physiol. 228(5) : 1615-1620. 1974.-The contractile responses of helically cut rat aortic strips to neurohypophyseal hormones and synthetic analogues in the presence and absence of 1.2 mM external magnesium ions ([Mg++].) was studied. These experiments demonstrate that 1) [Mg++], potentiates the contractile actions of a variety of neurohypophyseal peptides on vascular smooth muscle. 2) [Mg++], can alter both the ED& (i.e., hormone-receptor affinities) and intrinsic activities (or maximum contractile responses) of these molecules on vascular muscle. 3) The amino acid moieties in positions 1, 2, 3, and 8 of the vasopressin molecule interact, differentially, with [Mg++IO to produce contraction of vascular muscle. 4) The length of the side chain, and basicity, in position 8 of the vasopressin molecule are probably important in Mg potentiation in mammalian vascular muscle. 5) Interaction of Mg with an aromatic group in position 3 might be critical for potency of vasopressin hormones on mammalian vascular muscle; the EDso for oxytocin is not shifted to lower concentrations in the presence of [Mg++IO . Collectively, these data suggest that Mg probably acts at more than one site in vascular smooth muscle in the production of neurohypophyseal peptide-activated contractions. In addition, the present findings indicate that the Mg dependence of these peptides on at least one vascular muscle, rat aorta, is a direct function of the rat pressor potency of the molecules.
ing) of these hormones to their receptors rather than the intrinsic activity (or ability to induce the maximal response) of the molecules. ’ A preliminary in vitro study emplaoying four synthetic neurohypophyseal hormone analogues suggests that the magnesium-dependence of these peptides on at least one type of mammalian blood vessel, namely, rat aorta, may be directly rather than inversely related to the potency of the hormones (12). In addition, this latter study suggests that magnesium can alter the maximum contractile response (or intrinsic activity) of these hormones. In view of these latter, unexpected findings the present in vitro study, employing nine highly purified, synthetic neurohypophyseal hormones and analogues, was designed to determine 1) the exact relationship between external magnesium ([Mg++]J and hormone potency on rat aorta; 2) whether the presence alters hormone-receptor affinity, intrinsic acof [Mg++]* tivity, or both; and 3) whether a relationship exists between magnesium’s potentiating effect and the amino acid moieties in positions 1, 2, 3, and 8 of the vasopressin lnolecule on contractile activity in vascular smooth muscle.
vasopressin;
Thoracic aortas were obtained from male rats (W&tar strain, 2004QO g), cut helically into vascular strips (1.31.5 mm in width by 25 mm in length), and set up isometrically, in vitro, under a resting tension of 1.5 g, essentially as described previously (7). Only male rats were employed in these studies since sex and estrogenic hormones are known to afFect the reactivity of blood vessels to neurohypophyseal hormones (4, 6). All vascular strips were equilibrated for 2 h in muscle chambers containing KrebsRinger-bicarbonate solution (with 1.2 mM Mg++); the composition of the Krebs-Ringer-bicarbonate solution has been given previously (2). The Krebs-Ringer-bicarbonate solution was oxygenated continuously with a 95 % 02-5 % CO2 mixture and kept at 37°C (pH 7.2-7.5). One of a pair of vascular strips (cut from same aorta) was then exposed for 1 h to a Mg ++-free Krebs-Ringer-bicarbonate solution, while the other strip (with 1.2 mM Mg++) was incubated in normal Krebs-Ringer. The incubation media were routinely changed every 10-l 5 min as a precaution against interfering metabolites (9). Complete, cumulative log-doseresponse curves, similar to those described previously (2, 3,
potency mone muscle
oxytocin;
hormone
receptors;
of neurohypophyseal hormones; analogues; magnesium-calcium
rat aorta; rat pressor
neurohypophyseal interactions in
horvascular
IT IS KNOWN THAT magnesium (Mg++) ions can potentiate the contractile actions of neurohypophyseal hormones on all types of smooth muscle so far investigated ( 14, 16, 18-20, 22, 25, 29, 30, 3 1). Similar Mg++-neurohypophyseal hormone interactions have recently been observed on a variety of isolated vertebrate and human blood vessels (3, 11, 25-28). Several of these in vitro studies suggest that potentiation of structurally different neurohypophyseal hormones and analogues by Mg++ on different effector systems (e.g*, uterus, mammary gland, blood vessels) is inversely related to potency of the molecules and not to intrinsic differences between uterine smooth muscle, myo-epithelial, or vascular smooth muscle receptors, per se (14, 16, 18, 20, 25, 30). In this context, Somlyo, Woo, and Somlyo (28) have postulated that Mg++ ions primarily affect the affinity (or bind-
METHODS
1615
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1616
B. M.
7), were obtained for nine different synthetic neurohypophyseal hormone analogues. The synthetic neurohypophyseal hormones and analogues used were [8-argininejvasopressin (approximate assay by rat. pressor method = 400 IU/mg) ; [8-lysinelvasopressin (270 IU/mg) ; [3-isoleucine ,8-leucine]vasopressin (i.e., oxytocin) (5 IU/mg) ; l -Deamino - [8-argininelvasopressin (385 IU/mg); [8ornithinelvasopressin (360 IU/mg) ; [Z-phenylalanine , 8-lysinelvasopressin (57 IU/mg) ; [3 - isoleucine ,8 - ornithinelvasopressin (102 IU/mg) ; [2-phenylalanine , S-isoleucine ,8-ornithine] vasopressin (120 IU/mg) ; and lDeamino-[Z-phenylalanine ,8-argininelvasopressin (35 IU/ mg). All these peptides were free of chlorobutanol preservative. The peptides were suitably diluted with 0.5 % acetic acid, ampouled, and used as standards throughout the experiments. Further dilutions were made up fresh as necessary. The oxytocin (also preservative free) was obtained from Sandoz Ltd. in high potency (i.e., 430 IU/ml). The results for homone-receptor affinities are given in ED& (me& effective dose) (2, 7, 26, 31), while the intrinsic activities, or percent maximal responses, are all relative to [8-argininelvasopressin since the latter is the native rat pituitary hormone. The ED 50 concentration is used as an approximation of the relative potency of the structurally altered peptide (7, 30, 3 l)* The actual maximum developed tensions (in mg) are also reported. A total of 185 rats was utilized for the present study. RESULTS
Inj%.mce of extracellulurA@++ on cumulative dose-response curves to arginine-vasopressin and oxytacin on isolated rut aorta. Figure 1 demonstrates that the presence of 1.2 mM [Mg++J,
ALTURA
not only shifts the dose-response curve for [Args]vasopressin to the left of the Mg ++-free strips approximately sevenfold, but concomitantly increases the maximum contractile response almost fivefold. Figure 1 also demonstrates that although the maximum contractile response for oxytocin is likewise potentiated by the presence of [Mg++], approximately sevenfold, the dose-response curve for this hormone is not shifted to the left of the curve for the absence of The smaller [Mg++lo, as is the case for [Arg8]vasopressin. contractile responses seen in zero [MS++]. are probably not due to traces of Mg++, since the developed tensions for both [Arg 8]vasopressin and oxytocin were not significantly different from those obtained when 1 mM Nas-EDTA (ethylenediaminetetraacetic acid, sodium salt) was added to the zero [Mg++10 (five experiments each)* InjGence of 1.2 mA-4[Mg++]o on intrinsic activities of neurohypophyseal pep tides with amino acids substituted in positions I, 2, 3, and/or 8. Table 1 summarizes the results obtained for nine different neurohypophyseal peptides altered with respect to positions 1, 2, 3 and/or 8 of the vasopressin molecule. First, it should be noted that the presence of a physiologic concentration of Mg approximately equalizes the maximal contractile response to all nine peptides whereas in the absence of Mg the values range from as little as 6.9 to 387.6 mg. Second, these data indicate that although the presence of extracellular magnesium significantly enhances (potentiates) the maximal contractile responses of all nine peptides, the relative increase in the neurohypophyseal contractile response is somehow dependent on the chemical structure of the amino acid substituents in the synthesized peptide analogues. For example, I) the presence of an amino group in position l (i.e., [Argglvasopressin vs. l-De-
100 2
$ iT 51
m
1.2mM/L
M
Mg++- Free
Mg*
M
1.2mMiL
-
Mg++- Free
90-
2
8Q-
I Q1 .-C C .-
70-
0 4 z
60-
? z
50-
5 s
40-
vp
r
I Mg*+
oxy 3
NEUROHYPOPHYSEAt FIG, 1. Cumulative dose-response curves of [8-argininelvasopressin and oxytocin in Krebs-Ringer bicarbonate with and without 1.2 mM Mg++. e--e, vasopressin with added Mg++; O--Q vasopressin
HORMONE
(Molar
without [Mg++].; oxytocin without least 12 different
Cont.)
A--A, [Mg++].. rat aortic
oxytocin with added Each curve represents strips.
Mg++; A--A, an average of at
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MAGNESIUM,
NEUROHYPOPHYSEAL
HORMONES,
AND
VASCULAR
TABLE f.. hj%wnce of 1.2 mM [Mg++](, on neurohy~u~hys~~ jw;btide hormonal +@2ilies and maximum contractile res;Donsesin isolated rat aortic stri’s -__ -:-I----~~~ Rat 0 n-m [Mg+-+]o 1.2 ml1 [Mg++], pressor -_- -- --~~Vasopressin Analogue
Activity, W-w
Developed tension, m g
[Argsj
400
1-Dearnino[A&] [Orn 8]
385
KY s81J
270
[Phe2, Ile”, Orn *] [ Ile” , Orn 81
120
181.9 zt18.2 97.8 + 6.5 387.6 zt39.5 74.8 zt 2.7 6.9 It 0.9 179.4 zt17.7 240.4 zt23 -2 266.8 zt24.5 113.1 +13 .8
360
102
[Phe2,Lys8]
57
I -Deamino[ Phe2, Arg *] [ Ile” , Leu 81
35 5
E&o, x10-” M
1.9 zt 0.2 120.3 zt14.7 2.1 zt 0.2 22.0 xt 1.4 60.0 It 2.5 5.8 * 0.7 100.3 zt14.2 82.0 + 3.1 1400 g35.3
-----Values represent the means + SE strips. Only brie peptide was utilized METHODS) . * Significantly different absence of [hIg++](, (P < 0.00 1 >.
Developed tension, mg
--.
946.7 zt82.6* 903 * I3 *79.5* 954.2 +84.7* 850 *0 *70.5* 840.2 AZ81 .IP 939.6 zt80.6* 830.9 z/=71.7* 832.6 zt68.5* 813.7 *57 0s9*
.--
EDSO, x10-9 M
_.-.. -
0.28 It 0.02* 14.1 * 0.8* 0.21 Et 0.02* 3.7 It 0.2” 15.0 I/= 0 9* 7:6 + 0.6 33.0 zt 1.7* 25.1 It 1.3* 2600 +57.9*
-.-~
of at Xcast 10 different aortic on each rat aortic strip (see from values obtained in the
amino-[Arg S]vasopressin) decreases the relative degree of potentiation in maximal contractile response obtained in the presence of 1.2 mM 1-Mg++]. from approximately 1Ofold to 5-fold; 2) removal of the phenolic hydroxyl in Arg 8]vasopressin and position 2 (e-g., I -Deamino-[Phe2, [ Phe2, Lys &]vasopressin vs. 1-Deamino-[Arg S]vasopressin and [LysS]vasopressin) also decreases the relative degree of potentiation in maximal contractile response (i.e., 3-fold, 3.6-fold vs. 1O-fold, 1Z-fold) ; and 3) a decrease in basicity in position 8 relative to [Arg8]vasopressin (e-g+, [LysS]vasocan result in increases pressin, and [Ile3, Leus]vasopressin) in the relative degree of potentiation of maximal response when magnesium is present (i.e., approximately l2-fold, 7.5-fold vs. 5-fold for [Arg 8]vasopressin) ; [Orn”]vasopressin, however, appears to be an exception. In addition, the data in Table 1 indicate that major amino acid changes in the native hormone, (e.g., [Phe2, Ile3 , Orn Y]vasopressin) can bring about an extreme degree of potentiation of maximal response in the presence of [Mg++],, (i.e., approximately l30-fold). Injhence of 1.2 mM [Mg++]. on the ED;& (;hotenc_?l) of naurohypo;b~~seul peptides with amino acids subshtuted in positions I, 2, 3, and/or 8. The ED& in Table 1 suggest that the greater the rat pressor potency of the neurohypophyseal peptide, the greater is the shift of the cumulative doseresponse to the left in the presence of [Mg+‘],. For example, [ArgS]vasopressin has a rat pressor activity approximately equal to 400 U/ m g and its EDjo is shifted sevenfold in the presence of 1.2 mM [Mg++]., while the rat pressor activity of [Phe2, Lys 8]vasopressin is approximately 57 U/mg and its EDs0 is shifted only threefold in the presence of [MS++].. Molecules having primarily an oxytocic structural backbone, e.g., [He”, LeuX]and [Ile3, O&]vasopressin, fail, however, to demonstrate a lower EDjo in the presence of 1.2 mM [Mg++].. A plot of the data in Table 1 (i.e., the
1617
MUSCLE
ratio of the EDjD of the peptide without 1.2 mM Mg++ to that with [Mg++],, vs. rat pressor potency) clearly indicates that the degree of potentiation of a neurohypophyseal peptide on rat aortic smooth muscle by [Mg++], is a direct function of the rat pressor potency of the neurohypophyseal peptide molecule (see Fig. 2). In other terms, the more potent the neurohypophyseal peptide is in raising rat blood pressure, the more dependent it is on extracellular magnesium ions for inducing contraction in rat aorta. DISCUSSION
The presence of [Mg++]. appears to result in two difierent types of effects on vasopressin peptidc-induced contractions: 1) an increased afinity of all1 the molecules used in this study (shifts to lower EDjo peptide concentrations), irrespective of chemical structure, for their receptors or active sites in rat aortic smooth muscle; and 2) an increase in the maximal contractile response. Such data could, thus, be interpreted to indicate that [Mg++10 cannot only affect events at the peptide receptors in vascular smooth muscle (3, 1 I, 25-28) but beyond (e.g., increase in peptide-induced maxima1 contractile responses) the receptors as well (ll), e.g., on excitation, coupling, or the contractile machinery per se. This concept would thus differ somewhat from that previously proposed by other workers, who have indicated that [Mg++], probably only afFects events at the peptidereceptor sites in smooth muscle (14, 16, 18, 22, 25, 26, 28). It is of interest to note here that other workers have found that [Mg++], can also aKect both the affinity and intrinsic activity of some of these peptides on rat uterine smooth muscle (19, 30, 3 I). The present findings and those of the latter workers could thus be used to support the idea previously promulgated by our group that [Mg++], could potentiate neurohypophyscal peptides by acting at several sites in vascular smooth muscle (10, 11). One of these Mg sites is probably at the membrane (or receptor surface) (10, 11, 14, 28) since the potentiating action occurs rapidly and disappears quickly when [Mg++& is withdrawn from the medium. Mechanistically, Mg++ could be envisioned as difFerentially activating a conformational change at the receptor site and/or the cell membrane (10, 11, 14, 28); in other terms a better Yit” between the neurohypophyseal peptides and the receptor molecules would take place in the presence of [Mg++J, (14). In this context, Soloff and Swartz (23, 24) have clearly demonstrated that Mg++ enhances the binding of tritiated oxytotin to 20,000 X g particles of rat uterine and mammary tissue; presumably these components arc integral parts of the oxytocin-receptor systems in the rat uterine and myo-epithelial tissues. A second site for Mg action appears to be intracellular in location (10, 11, 17a). The increased maximal contractile response of vertebrate vascular smooth muscle to neurohypophyseal peptides in the presence of [Mg++], (3, 11, 26, 27), as well as that of rat uterine smooth muscle (19, 30, 3 l), may be brought about by a displacement of calcium ions from intracellular sites. Others and ourselves have previwhich could be inously presented data, albeit indirect, 1 Oxytocin and this generalization.
a derivative
of oxytocin,
i.e.,
[Urn8]oxytocin,
do not
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1618
B. M, ALTURA +RN~]-VP
tl Ol
fiLE3,LEU8]-VP
I 50
+LE~,oRN~]-VP
1
100
I
150
I
200
I
250
I
300
I
350
I
400
1
450
RAT PRESSOR POTENCY (IUhicromoIe) FIG. 2. Potentiation in vitro of contractile action of neurohypophyseal hormones and analogues modified in positions 1, 2, 3, and 8 by magnesium ions on rat aortic strips. Ordinate: ratio of potencies
(i.e., EI&‘s) in presence and absence of 1.2 mM Mg++. Abscissa: ra pressor potencies (IU,$moI). Each value represents results of at leas 20 different paired aortic strips.
terpreted as support for the latter concept (10, 11, 17a, 28b). For example, although different vasotropic agents are known to induce contraction of smooth muscle via different (inreceptor systems, it is believed I) that all stimulants eluding neurohypophyseal peptides) require calcium ions for excitation-contraction coupling, and 2) the source of activator calcium may be extracellular or intracellular in nature, depending upon stimulant (15). The fact that [Mg++], has been shown to I) alter the binding of calcium ions in vascular (10, 11, 28b) as well as other types of smooth muscle (15a, %a), and 2) enhance the maximal contractile response of depolarized rat aorta to [Ca++]. (11) supports the idea that Mg ions can compete with Ca+f for certain membrane and intracellular sites. A freeing of intracellular ionized Ca for interaction with actomyosin could account for the increased maximal contractile responses observed in the presence of extracellular Mg. An alternative likely and contributing mechanism is that more actomyosin is activated by the presence of Mg++ @la). It has recently been suggested by Somlyo and Somlyo (25) that Mg may potentiate neurohypophyseal hormones by making available additional receptors for these peptides in the effector smooth muscle cells. Although the present study does not rule in or out this possibility, there are some indirect experiments in the recent literature that permit such a concept to be questioned. For example, Mg has been shown to potentiate, in addition to neurohypophyseal peptides and- certain amines (10, 11, 17a), barium-induced muscle (10 1 11) 1 contractions as well in aortic smooth barium being a vasoact ive substance that does not exert effects through a specific receptor. One would, therefore, be hard pressed to explain the potentiation of barium-
induced responses on the basis of magnesium making additional LLhormone receptors” available. A more likely explanation of Mg potentiation of barium contractions would be that Mg facilitates the translocation of a specific cellular Ca fraction necessary for the response to barium (10, 11). It must, however, be pointed out that the barium-induced contracti .ons are not associated with a shift in the doseresponse curve (EDso). Therefore, one must entertain the possibility that the two forms of potentiation (i.e., neurohypophyseal peptide and barium) may be mediated by different mechanisms. In the present study, an attempt has been made to determine whether the free amino group, OH group, aromatic group (i.e., phenylalanine), and basicity (and length of side chain) in positions 1, 2, 3, and 8, respectively, of the neurohypophyseal hormone structure are important in control of vasopressin potency by [Mg++10 in mammalian arterial muscle. Our findings indicate that the most basic peptide, i.e., [8-arginine]vasopressin, with arginine having a pk3 = 12.48, has its EDso with [Mg++J, shifted 6.8-fold to the left of that in the absence of [Mg++]., while [8-ornithinejvasopressin (with ornithine having a pka = 10.76) exhibits a much greater degree of potentiation in the presence of Mg, viz. lo-fold. These results tend to suggest that the length of the side chain in position 8 of the vasopressin molecule may be quite important in Mg potentiation in mammalian vascular muscle; i.e., ornithine has a shorter side chain than arginine* Our data with [8-lysine]vasopressin, with lysine having a pka = 10.28, but a side-chain length equivalent to that of arginine, could be used to support this tenet; the potentiation of lysine-vasopressin by Mg is approximately equivalent to that exhibited by
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MAGNESIUM,
NEUROHYPOPHYSEAL
HORMONES,
AND
VASCULAR
arginine-vasopressin, i.e., 6.9-fold (on the basis of ED&). In addition, these data suggest that although potentiation of neurohypophyseal peptides by Mg in mammalian vascular muscle may be somewhat dependent upon basicity in position 8 (e.g., [3-isoleucine, 8-leucine]vasopressin has a neutra1 molecule in position 8 and fails to exhibit potency shifts with Mg; i.e., the EDso is not shifted to lower conbasicity in this position centrations (Fig. I)), maximizing does not result in maximal contractile potency, e-g., compared to [8-ornithinelvasopressin. The data with l-Deamino-[8-argininelvasopressin indicate that 1.2 mM Mg shifts the ED 50 of this molecule almost ninefold; this is thus greater than the Mg potentiation that is seen with [8-argininelvasopressin. These findings tend to suggest that, at least with respect to rat aortic smooth muscle: a) the terminal, free amino group may not be important for Mg binding of the vasopressin hormone to its receptor; and b) the vasopressin molecule may actually interact (or bind more strongly) to Mg when a hydrogen is present rather than a free NH2 group. In addition, the present data suggest that although the phenolic group in position 2 is not critical for Mg-induced potentiation, the presence of an OH group in this position does enhance the interaction with Mg ions; e-g., although [Z-phenylalanine, 8-lysinelvasopressin and 1-Deamino-[Z-phenylalanine ,8-arginine] vasopressin are potentiated threefold each by [Mg++], (Fig. 2), [8-lysine] - and 1-Deamino-[8-argininelvasopressin are potentiated seven- and ninefold, respectively, by [Mg++], (Fig. 2) The findings with the substitution of isoleucine for phenylalanine in position 3 of [8-ornithine]vasopressin are of considerable interest. Interestingly, the EDso of the resultan t molecule, viz., [3-isoleucine, 8-ornithinelvasopressin is not changed by [Mg++]., quite unlike the tenfold potentiation by Mg of [8-ornithinelvasopressin (Fig. 2). These findings indicate that the interaction of Mg with an aromatic group (or phenylalanine) might be critical for potency of vasopressin contractions on mammalian vascuXar smooth muscle. Our findings concerning position 3 and Mg potentiation on vascular smooth muscle are thus quite diRerent from those reported for this position in uterine smooth muscle (30) It is rather interesting to note that of the three vasopressin analogues (or oxytocin derivatives) used here with isoleucine in position 3 (see Table 1 and Fig. 2), only one of them, viz., [2-phenylalanine , 3-isoleucine , &ornithine]vasopressin, has its ED 50 shifted by Mg (Fig. 2) ; and even here the displacement is only fourfold. These findings not only strengthen the notion that an aromatic group in position 3 of these hormones is important for Mg control of vasopressin potency (Le., to produce a shift in EDjo) on rat arterial smooth muscle (and maybe somatic vascular muscle in general), but could be used to suggest that there are two distinct, separate receptors for vasopressin and oxytocin in mammalian vascular smooth muscle. Several of the fmdings in this paper and in the literature could be utilized to buttress this tenet: I) oxytocin is an extremely weak contractile substance (e.g., exhibits highest ED& and lowest intrinsic activities or efficacies), not only on rat aortas described here, but on every other type of somatic microand macrovascular smooth muscle preparation so far l
1619
MUSCLE
examined findings).
in a variety of mammals (2, 5, 8, 25, and present 2) Although potency of oxytocin is not potentiated by 1.2 mM [MS++]. (i.e., shift the EDSO to lower concentrations) on rat aortic strips, both arginine-vasopressin and lysine-vasopressin ED 50’s are strongly potentiated by [Mg++],. This is thus different from that seen on rat uterine smooth muscle where vasopressin is only slightly more potentiated by Mg than is oxytocin (30). 3) On highly specialized vascular smooth muscle, such as that found in human umbilical arteries and veins, oxytocin exhibits not only a much greater afinity for its receptor site than does arginine-vasopressin but a much greater intrinsic activity as well (13) ; this is thus the reverse of that seen for these two posterior pituitary hormones on mammalian somatic blood vessels. 4) Arginine-vasopressin (preservative free) and its analogues have been shown to relax bovine coronary arteries (I), while oxytocin (preservative free) fails to induce similar action on these vessels (unpublished observations). 5) Low concentrations of synthetic oxytocin can induce potent relaxations of a variety of intact mesenteric microvessels in male rats, but vasopressin can only elicit contractile responses on these same vessels (4). Although these findings, collectively, do suggest that there may be separate receptor sites for oxytocin and vasopressin in mammalian vascular muscle, further work will be required before this hypothesis can be accepted. Specific competitive antagonists of vasopressin and oxytocin (17, 21) will be required to discern whether there are, indeed, two separate receptors for vasopressin and oxytocin in mammalian vascular muscle. The present study, in denronstrating that potentiation of nine structurally different neurohypophyseal hormones and analogues by external Mg ions is a direct function of the rat pressor potency of the molecules, indicates that Mgneurohypophyseal hormone interactions in vascular muscle may be somewhat different from those taking place in uterine smooth muscle. All the studies done so far with uterine smooth muscle indicate that potentiation of neurohypophyseal peptides by Mg++ is inversely related to the uterotonic potency of these molecules, not directly (14, 16, 18, 20, 25, 30, 3 1). Th is is probably due to intrinsic differences between the uterine smooth muscle and vascular muscle receptors for these posterior pituitary hormones. Such fundamental differences could prove useful in the characterization of the receptors for these hormones in blood vessels. I am grateful for the excellent technical assistance provided in these studies by Marjorie K. Nicodemus and Charles F. Reich. I am indebted to the late Dr. R. Bircher and Dr. B. Berde of Sandoz Ltd., as well as to Dr. R. Walter, Mt. Sinai School of Medicine, for generously supplying the synthetic peptides used in these studies. This study was supported in part by Public Health Service Research Grant HE-18015 and Grant HL-12462 from the National Heart and Lung Institute. Part of this study was performed during the tenure of B, M. Altura of a Research Career Development Award from the National Institutes of Health, Public Health Service. Requests for reprints should be sent to: B. M. Altura, Dept. of Physiology, Box 31, State University of New York, Downstate Medical Center, Brooklyn, N. Y. 11203. Received
for publication
14 June
1974.
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B.
1620
M.
ALTURA
REFERENCES 1. ALTURA, B. M. Differential actions of polypeptides and other drugs on coronary inflow vessels. Am. Heart J. 72 : 709-711, 1966. 2. ALTURA, B. M. Significance of amino acid residues in vasopressin on contraction in vascular muscle. Am. J. Physiol. 219: 222-229, 1970. 3. ALTURA, B, M. Influence of magnesium and cysteine on vasopressin-induced contractions in various canine blood vessels. Ex/wrientia 26 : 1089-1090, 1970. 4. RLTURA, B. M. Chemical and humoral regulation of blood flow through the precapillary sphincter. Microvascular Res. 3 : 36 l-384, 1971. 5. ~ZLTURA, B. M. Significance and interaction of the amino acid residues in positions 1, 2, 3 and 8 of vasopressins on contractile activity in vascular smooth muscle. In: Chemistry and Biology of P@tides, edited by J. Meienhofer. Ann Arbor : Ann Arbor Science, 1972, p. 441-447. 6. ALT~RA, B. h& S ex and sex hormones as factors influencing the responsiveness of arterioles to catecholamines and neurohypophyseal hormones. flficroaascular Res. 6 : 117, 1973. 7. ALTURA, B. M. Significance of amino acid residues in position 8 of vasopressin on contraction of rat blood vessels. Proc. Sot, &ptl. Biol. k&d. 142: 1104-1110, 1973. 8. ALTURA, B. M. Selective microvascular constrictor actions of some neurohypophyseal peptides. European J, Pharmacol. 24: 49-60, 1973. 9. ALTWRA, B. M., AND B. T. ALTURA, Differential effects of substrate depletion on drug-induced contractions of rabbit aorta. Am. J. Physiol. 219: 1698-1705, 1970. 10. ALTURA, B. M., AND B. T. ALTURA. Influence of magnesium on drug-induced contractions and ion content in rabbit aorta. Am. J. Physiol. 220 : 938-944, 1971. 11. RLTURA, B. M., AND B. T. ALTURA. Magnesium and contraction of arterial smooth muscle. Microuascular Res. 7 : 145-155, 1974. 12. ALTURA, B. M., AND R. W. BURTON. Structure-activity relationships of neurohypophyseal hormones on rat arterial and arteriolar smooth muscle : relationship of magnesium to contractile activity. Federation Proc. 31. : 542, 1972. 13. ALTURA, B. M., D. MALAVIYA, C. F. REICH, AND L,. R. ORKIN. Effects of vasoactive agents on isolated human umbilical arteries and veins. Am. J. Physiol. 222: 345-355, 1972. 14. BENTLEY, P. J. The potentiating action of magnesium and manganese on the oxytocic effect of some oxytocin analogues. ,J. Endocrinol. 32 : 2 15-222, 1965. 15. BOHR, D. F. Vascular smooth muscle updated. Circulation Xes. 32: 665-672, 1973. lSa.BoLToN,T. C., K.A. BERKICH, c. P. BIANCHI, AND T.J. ROHNER. Effects of Ca and Mg on contractile responses of dog detrussor muscle to bethanechol. Federation Proc. 33 : 540, 1974. 16. CHAN, We Y., AND N. KELLEY. A pharmacologic analysis on the significance of the chemical functional groups of oxytocin to its oxytocic activity and on the effect of magnesium on the in vitro and in vivo oxytocic activity of neurohypophysial hormones. J. Pharmacol. Exptl. Therap. 156 : 150-158, 1967. 17. DYCKES, D. F., J. J* NESTOR, JR., M. F. FERGER, AND V. DU VIGNEAUD. [I-D-M ercapto-@ , P-diethylpropionic acid] -8-lysinevasopressin, a potent i n h’b’t1 r or of 8-lysine-vasopressin and of oxytocin. J, Med. Chem. 17 : 250-252, 1974. ~~ZLJURKEVICS, H. A., AND 0. CARRIER, *JR. Effect of magnesium on
responses of aortas Am. J. Physiol. 225
from normal and reserpine-treated rabbits. : 1479-1485, 1973, 18, KREJ~, I., AND POLAEEK. Effect of magnesium on the action of oxytocin and a group of analogues on the uterus in vitro. European J. Pharmacol. 2: 393-398, 1968. 19. KREJtii, I., I. POLL~EK, B. KUPKOVA, AND J. IIUDINGER. Doseresponse analysis of the action of some oxytocin analogues on the isolated uterus: the effect of ions. In: Oxytocin, Vasopressin and their Strucfurd Analogties, edited by IJ. Rudinger. London: Pergamon, 1964, p. 117-123. 20. MUNSICK, R. A. Effect of magnesium ion on the response of the rat uterus to neurohypophysial hormones and analogues. Endocrinology 66:451-462, 1960. 21. ICUDINGER, ,J., AND I. KREJ% . Lxntagonists of the neurohypophysial hormones. In : Handbook of Exferimental Pharmacology. Neurohypophysial Hormones and Similar PolypeptidEs, edited by B. Berde. Heidelberg: Springer-Verlag, 1968, vol, 23, p. 748-801. 21a.KussELq W. E. Insolubilization and activation of arterial actomyosin by bivalent cations. European J. Biochem. 33: 549-566, 1973. 22. SCHILD, 0. H. The effect of metals on the S-S polypeptide receptor in depolarized rat uterus. Bit. J. Pharmacol. 36 : 3 29-349, 1969. 23. SOLOFF, h4. S., AND T. L. SWARTZ. Characterization of a proposed oxytocin receptor in rat mammary gland. ,I. Hiol. Chem. 248:6471-6478, 1973. 24. SOLOFF, M. S., AND T. L. SWARTZ. Characterization of a proposed oxytocin receptor in the uterus of the rat and sow. J. Biol. Gem. 249: 1376-1381, 1974. 25. SOMLYO, A. P., AND A. V. SOMLYO. Vascular smooth muscle. II. Pharmacology of normal and hypertensive vessels. Pharmacol. Rev. 22: 249-353, 1970. 26. SOMLYO, A. V., AND A. P. SOMLYO. Vasotocin-magnesium interaction in vascular smooth muscle of the hagfish (Eftatrstu stoutii). Camp. Biochem. Physiol. 24 : 267-270, 1968. 27, SOMLYO, A. P., A. V. SOMLYO, AND C.-Y. Woo. Neurohypophysial peptide interaction with magnesium in avian vascular smooth muscle. J, Physiol., London 192 : 657-668, 1967. 28. SOMLYO, A. V., C. Woo, AND ,4. P. SOMLYO. Effect of magnesium on posterior pituitary hormone action on vascular smooth muscle. Am. J. Physiol. 2 10 : 705-7 14, 1966. 28a,SPARROW, M. P. Interaction of 28Mg with Ca and K in the smooth muscle of guinea-pig taenia coli. J. l’hysiol., London 205 : 19-38, 1969. 28b.TENNEq T. E., JR., AND 0. CARRIER, *JR. Further studies on the interaction of potassium and magnesium in vascular muscle. Federation Proc. 33 : 435, 1974. 29. VAN DYKE, H. B., AND A. B. HASTINGS. The response of smooth muscle in different ionic environments. Am. J. Phvsiol. 83: 563577, 1928. 30. WALTER, R., B. h/z. DUBOIS, AND I. 1,. SCHWARTZ. Biological significance of the amino acid residue in position 3 of neurohypophyseal hormones and the effect of magnesium on their uterotonic action. Endocrinology 83 : 979-983, 1968. 31. YAMANAKA, T., S. HASE, S. SAKAKIBARA, I. L. SCHWARTZ, B. M. DUBOIS, AND R. WALTER. Crystalline deamino-dicarba-oxytocin. Preparation and some pharmacological properties. iMo2. Pharmacol. 6: 474-480, 1970.
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Magnesium-neurohypophyseal in contraction
hormone
of vascular
smooth
interactions
muscle
BURTON M. ALTURA Departments of Anesthesiology and Physiology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, and Department of Physiology, State University of .A New York Downstate Medical Center, Brooklyn, New York 11203
ALTURA, BURTON R/I. Mag~sium-neurohypuphyseal hormone interactions in contraction of vascular smooth muscle. Am. J. Physiol. 228(5) : 1615-1620. 1974.-The contractile responses of helically cut rat aortic strips to neurohypophyseal hormones and synthetic analogues in the presence and absence of 1.2 mM external magnesium ions ([Mg++].) was studied. These experiments demonstrate that 1) [Mg++], potentiates the contractile actions of a variety of neurohypophyseal peptides on vascular smooth muscle. 2) [Mg++], can alter both the ED& (i.e., hormone-receptor affinities) and intrinsic activities (or maximum contractile responses) of these molecules on vascular muscle. 3) The amino acid moieties in positions 1, 2, 3, and 8 of the vasopressin molecule interact, differentially, with [Mg++IO to produce contraction of vascular muscle. 4) The length of the side chain, and basicity, in position 8 of the vasopressin molecule are probably important in Mg potentiation in mammalian vascular muscle. 5) Interaction of Mg with an aromatic group in position 3 might be critical for potency of vasopressin hormones on mammalian vascular muscle; the EDso for oxytocin is not shifted to lower concentrations in the presence of [Mg++IO . Collectively, these data suggest that Mg probably acts at more than one site in vascular smooth muscle in the production of neurohypophyseal peptide-activated contractions. In addition, the present findings indicate that the Mg dependence of these peptides on at least one vascular muscle, rat aorta, is a direct function of the rat pressor potency of the molecules.
ing) of these hormones to their receptors rather than the intrinsic activity (or ability to induce the maximal response) of the molecules. ’ A preliminary in vitro study emplaoying four synthetic neurohypophyseal hormone analogues suggests that the magnesium-dependence of these peptides on at least one type of mammalian blood vessel, namely, rat aorta, may be directly rather than inversely related to the potency of the hormones (12). In addition, this latter study suggests that magnesium can alter the maximum contractile response (or intrinsic activity) of these hormones. In view of these latter, unexpected findings the present in vitro study, employing nine highly purified, synthetic neurohypophyseal hormones and analogues, was designed to determine 1) the exact relationship between external magnesium ([Mg++]J and hormone potency on rat aorta; 2) whether the presence alters hormone-receptor affinity, intrinsic acof [Mg++]* tivity, or both; and 3) whether a relationship exists between magnesium’s potentiating effect and the amino acid moieties in positions 1, 2, 3, and 8 of the vasopressin lnolecule on contractile activity in vascular smooth muscle.
vasopressin;
Thoracic aortas were obtained from male rats (W&tar strain, 2004QO g), cut helically into vascular strips (1.31.5 mm in width by 25 mm in length), and set up isometrically, in vitro, under a resting tension of 1.5 g, essentially as described previously (7). Only male rats were employed in these studies since sex and estrogenic hormones are known to afFect the reactivity of blood vessels to neurohypophyseal hormones (4, 6). All vascular strips were equilibrated for 2 h in muscle chambers containing KrebsRinger-bicarbonate solution (with 1.2 mM Mg++); the composition of the Krebs-Ringer-bicarbonate solution has been given previously (2). The Krebs-Ringer-bicarbonate solution was oxygenated continuously with a 95 % 02-5 % CO2 mixture and kept at 37°C (pH 7.2-7.5). One of a pair of vascular strips (cut from same aorta) was then exposed for 1 h to a Mg ++-free Krebs-Ringer-bicarbonate solution, while the other strip (with 1.2 mM Mg++) was incubated in normal Krebs-Ringer. The incubation media were routinely changed every 10-l 5 min as a precaution against interfering metabolites (9). Complete, cumulative log-doseresponse curves, similar to those described previously (2, 3,
potency mone muscle
oxytocin;
hormone
receptors;
of neurohypophyseal hormones; analogues; magnesium-calcium
rat aorta; rat pressor
neurohypophyseal interactions in
horvascular
IT IS KNOWN THAT magnesium (Mg++) ions can potentiate the contractile actions of neurohypophyseal hormones on all types of smooth muscle so far investigated ( 14, 16, 18-20, 22, 25, 29, 30, 3 1). Similar Mg++-neurohypophyseal hormone interactions have recently been observed on a variety of isolated vertebrate and human blood vessels (3, 11, 25-28). Several of these in vitro studies suggest that potentiation of structurally different neurohypophyseal hormones and analogues by Mg++ on different effector systems (e.g*, uterus, mammary gland, blood vessels) is inversely related to potency of the molecules and not to intrinsic differences between uterine smooth muscle, myo-epithelial, or vascular smooth muscle receptors, per se (14, 16, 18, 20, 25, 30). In this context, Somlyo, Woo, and Somlyo (28) have postulated that Mg++ ions primarily affect the affinity (or bind-
METHODS
1615
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1616
B. M.
7), were obtained for nine different synthetic neurohypophyseal hormone analogues. The synthetic neurohypophyseal hormones and analogues used were [8-argininejvasopressin (approximate assay by rat. pressor method = 400 IU/mg) ; [8-lysinelvasopressin (270 IU/mg) ; [3-isoleucine ,8-leucine]vasopressin (i.e., oxytocin) (5 IU/mg) ; l -Deamino - [8-argininelvasopressin (385 IU/mg); [8ornithinelvasopressin (360 IU/mg) ; [Z-phenylalanine , 8-lysinelvasopressin (57 IU/mg) ; [3 - isoleucine ,8 - ornithinelvasopressin (102 IU/mg) ; [2-phenylalanine , S-isoleucine ,8-ornithine] vasopressin (120 IU/mg) ; and lDeamino-[Z-phenylalanine ,8-argininelvasopressin (35 IU/ mg). All these peptides were free of chlorobutanol preservative. The peptides were suitably diluted with 0.5 % acetic acid, ampouled, and used as standards throughout the experiments. Further dilutions were made up fresh as necessary. The oxytocin (also preservative free) was obtained from Sandoz Ltd. in high potency (i.e., 430 IU/ml). The results for homone-receptor affinities are given in ED& (me& effective dose) (2, 7, 26, 31), while the intrinsic activities, or percent maximal responses, are all relative to [8-argininelvasopressin since the latter is the native rat pituitary hormone. The ED 50 concentration is used as an approximation of the relative potency of the structurally altered peptide (7, 30, 3 l)* The actual maximum developed tensions (in mg) are also reported. A total of 185 rats was utilized for the present study. RESULTS
Inj%.mce of extracellulurA@++ on cumulative dose-response curves to arginine-vasopressin and oxytacin on isolated rut aorta. Figure 1 demonstrates that the presence of 1.2 mM [Mg++J,
ALTURA
not only shifts the dose-response curve for [Args]vasopressin to the left of the Mg ++-free strips approximately sevenfold, but concomitantly increases the maximum contractile response almost fivefold. Figure 1 also demonstrates that although the maximum contractile response for oxytocin is likewise potentiated by the presence of [Mg++], approximately sevenfold, the dose-response curve for this hormone is not shifted to the left of the curve for the absence of The smaller [Mg++lo, as is the case for [Arg8]vasopressin. contractile responses seen in zero [MS++]. are probably not due to traces of Mg++, since the developed tensions for both [Arg 8]vasopressin and oxytocin were not significantly different from those obtained when 1 mM Nas-EDTA (ethylenediaminetetraacetic acid, sodium salt) was added to the zero [Mg++10 (five experiments each)* InjGence of 1.2 mA-4[Mg++]o on intrinsic activities of neurohypophyseal pep tides with amino acids substituted in positions I, 2, 3, and/or 8. Table 1 summarizes the results obtained for nine different neurohypophyseal peptides altered with respect to positions 1, 2, 3 and/or 8 of the vasopressin molecule. First, it should be noted that the presence of a physiologic concentration of Mg approximately equalizes the maximal contractile response to all nine peptides whereas in the absence of Mg the values range from as little as 6.9 to 387.6 mg. Second, these data indicate that although the presence of extracellular magnesium significantly enhances (potentiates) the maximal contractile responses of all nine peptides, the relative increase in the neurohypophyseal contractile response is somehow dependent on the chemical structure of the amino acid substituents in the synthesized peptide analogues. For example, I) the presence of an amino group in position l (i.e., [Argglvasopressin vs. l-De-
100 2
$ iT 51
m
1.2mM/L
M
Mg++- Free
Mg*
M
1.2mMiL
-
Mg++- Free
90-
2
8Q-
I Q1 .-C C .-
70-
0 4 z
60-
? z
50-
5 s
40-
vp
r
I Mg*+
oxy 3
NEUROHYPOPHYSEAt FIG, 1. Cumulative dose-response curves of [8-argininelvasopressin and oxytocin in Krebs-Ringer bicarbonate with and without 1.2 mM Mg++. e--e, vasopressin with added Mg++; O--Q vasopressin
HORMONE
(Molar
without [Mg++].; oxytocin without least 12 different
Cont.)
A--A, [Mg++].. rat aortic
oxytocin with added Each curve represents strips.
Mg++; A--A, an average of at
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MAGNESIUM,
NEUROHYPOPHYSEAL
HORMONES,
AND
VASCULAR
TABLE f.. hj%wnce of 1.2 mM [Mg++](, on neurohy~u~hys~~ jw;btide hormonal +@2ilies and maximum contractile res;Donsesin isolated rat aortic stri’s -__ -:-I----~~~ Rat 0 n-m [Mg+-+]o 1.2 ml1 [Mg++], pressor -_- -- --~~Vasopressin Analogue
Activity, W-w
Developed tension, m g
[Argsj
400
1-Dearnino[A&] [Orn 8]
385
KY s81J
270
[Phe2, Ile”, Orn *] [ Ile” , Orn 81
120
181.9 zt18.2 97.8 + 6.5 387.6 zt39.5 74.8 zt 2.7 6.9 It 0.9 179.4 zt17.7 240.4 zt23 -2 266.8 zt24.5 113.1 +13 .8
360
102
[Phe2,Lys8]
57
I -Deamino[ Phe2, Arg *] [ Ile” , Leu 81
35 5
E&o, x10-” M
1.9 zt 0.2 120.3 zt14.7 2.1 zt 0.2 22.0 xt 1.4 60.0 It 2.5 5.8 * 0.7 100.3 zt14.2 82.0 + 3.1 1400 g35.3
-----Values represent the means + SE strips. Only brie peptide was utilized METHODS) . * Significantly different absence of [hIg++](, (P < 0.00 1 >.
Developed tension, mg
--.
946.7 zt82.6* 903 * I3 *79.5* 954.2 +84.7* 850 *0 *70.5* 840.2 AZ81 .IP 939.6 zt80.6* 830.9 z/=71.7* 832.6 zt68.5* 813.7 *57 0s9*
.--
EDSO, x10-9 M
_.-.. -
0.28 It 0.02* 14.1 * 0.8* 0.21 Et 0.02* 3.7 It 0.2” 15.0 I/= 0 9* 7:6 + 0.6 33.0 zt 1.7* 25.1 It 1.3* 2600 +57.9*
-.-~
of at Xcast 10 different aortic on each rat aortic strip (see from values obtained in the
amino-[Arg S]vasopressin) decreases the relative degree of potentiation in maximal contractile response obtained in the presence of 1.2 mM 1-Mg++]. from approximately 1Ofold to 5-fold; 2) removal of the phenolic hydroxyl in Arg 8]vasopressin and position 2 (e-g., I -Deamino-[Phe2, [ Phe2, Lys &]vasopressin vs. 1-Deamino-[Arg S]vasopressin and [LysS]vasopressin) also decreases the relative degree of potentiation in maximal contractile response (i.e., 3-fold, 3.6-fold vs. 1O-fold, 1Z-fold) ; and 3) a decrease in basicity in position 8 relative to [Arg8]vasopressin (e-g+, [LysS]vasocan result in increases pressin, and [Ile3, Leus]vasopressin) in the relative degree of potentiation of maximal response when magnesium is present (i.e., approximately l2-fold, 7.5-fold vs. 5-fold for [Arg 8]vasopressin) ; [Orn”]vasopressin, however, appears to be an exception. In addition, the data in Table 1 indicate that major amino acid changes in the native hormone, (e.g., [Phe2, Ile3 , Orn Y]vasopressin) can bring about an extreme degree of potentiation of maximal response in the presence of [Mg++],, (i.e., approximately l30-fold). Injhence of 1.2 mM [Mg++]. on the ED;& (;hotenc_?l) of naurohypo;b~~seul peptides with amino acids subshtuted in positions I, 2, 3, and/or 8. The ED& in Table 1 suggest that the greater the rat pressor potency of the neurohypophyseal peptide, the greater is the shift of the cumulative doseresponse to the left in the presence of [Mg+‘],. For example, [ArgS]vasopressin has a rat pressor activity approximately equal to 400 U/ m g and its EDjo is shifted sevenfold in the presence of 1.2 mM [Mg++]., while the rat pressor activity of [Phe2, Lys 8]vasopressin is approximately 57 U/mg and its EDs0 is shifted only threefold in the presence of [MS++].. Molecules having primarily an oxytocic structural backbone, e.g., [He”, LeuX]and [Ile3, O&]vasopressin, fail, however, to demonstrate a lower EDjo in the presence of 1.2 mM [Mg++].. A plot of the data in Table 1 (i.e., the
1617
MUSCLE
ratio of the EDjD of the peptide without 1.2 mM Mg++ to that with [Mg++],, vs. rat pressor potency) clearly indicates that the degree of potentiation of a neurohypophyseal peptide on rat aortic smooth muscle by [Mg++], is a direct function of the rat pressor potency of the neurohypophyseal peptide molecule (see Fig. 2). In other terms, the more potent the neurohypophyseal peptide is in raising rat blood pressure, the more dependent it is on extracellular magnesium ions for inducing contraction in rat aorta. DISCUSSION
The presence of [Mg++]. appears to result in two difierent types of effects on vasopressin peptidc-induced contractions: 1) an increased afinity of all1 the molecules used in this study (shifts to lower EDjo peptide concentrations), irrespective of chemical structure, for their receptors or active sites in rat aortic smooth muscle; and 2) an increase in the maximal contractile response. Such data could, thus, be interpreted to indicate that [Mg++10 cannot only affect events at the peptide receptors in vascular smooth muscle (3, 1 I, 25-28) but beyond (e.g., increase in peptide-induced maxima1 contractile responses) the receptors as well (ll), e.g., on excitation, coupling, or the contractile machinery per se. This concept would thus differ somewhat from that previously proposed by other workers, who have indicated that [Mg++], probably only afFects events at the peptidereceptor sites in smooth muscle (14, 16, 18, 22, 25, 26, 28). It is of interest to note here that other workers have found that [Mg++], can also aKect both the affinity and intrinsic activity of some of these peptides on rat uterine smooth muscle (19, 30, 3 I). The present findings and those of the latter workers could thus be used to support the idea previously promulgated by our group that [Mg++], could potentiate neurohypophyscal peptides by acting at several sites in vascular smooth muscle (10, 11). One of these Mg sites is probably at the membrane (or receptor surface) (10, 11, 14, 28) since the potentiating action occurs rapidly and disappears quickly when [Mg++& is withdrawn from the medium. Mechanistically, Mg++ could be envisioned as difFerentially activating a conformational change at the receptor site and/or the cell membrane (10, 11, 14, 28); in other terms a better Yit” between the neurohypophyseal peptides and the receptor molecules would take place in the presence of [Mg++J, (14). In this context, Soloff and Swartz (23, 24) have clearly demonstrated that Mg++ enhances the binding of tritiated oxytotin to 20,000 X g particles of rat uterine and mammary tissue; presumably these components arc integral parts of the oxytocin-receptor systems in the rat uterine and myo-epithelial tissues. A second site for Mg action appears to be intracellular in location (10, 11, 17a). The increased maximal contractile response of vertebrate vascular smooth muscle to neurohypophyseal peptides in the presence of [Mg++], (3, 11, 26, 27), as well as that of rat uterine smooth muscle (19, 30, 3 l), may be brought about by a displacement of calcium ions from intracellular sites. Others and ourselves have previwhich could be inously presented data, albeit indirect, 1 Oxytocin and this generalization.
a derivative
of oxytocin,
i.e.,
[Urn8]oxytocin,
do not
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1618
B. M, ALTURA +RN~]-VP
tl Ol
fiLE3,LEU8]-VP
I 50
+LE~,oRN~]-VP
1
100
I
150
I
200
I
250
I
300
I
350
I
400
1
450
RAT PRESSOR POTENCY (IUhicromoIe) FIG. 2. Potentiation in vitro of contractile action of neurohypophyseal hormones and analogues modified in positions 1, 2, 3, and 8 by magnesium ions on rat aortic strips. Ordinate: ratio of potencies
(i.e., EI&‘s) in presence and absence of 1.2 mM Mg++. Abscissa: ra pressor potencies (IU,$moI). Each value represents results of at leas 20 different paired aortic strips.
terpreted as support for the latter concept (10, 11, 17a, 28b). For example, although different vasotropic agents are known to induce contraction of smooth muscle via different (inreceptor systems, it is believed I) that all stimulants eluding neurohypophyseal peptides) require calcium ions for excitation-contraction coupling, and 2) the source of activator calcium may be extracellular or intracellular in nature, depending upon stimulant (15). The fact that [Mg++], has been shown to I) alter the binding of calcium ions in vascular (10, 11, 28b) as well as other types of smooth muscle (15a, %a), and 2) enhance the maximal contractile response of depolarized rat aorta to [Ca++]. (11) supports the idea that Mg ions can compete with Ca+f for certain membrane and intracellular sites. A freeing of intracellular ionized Ca for interaction with actomyosin could account for the increased maximal contractile responses observed in the presence of extracellular Mg. An alternative likely and contributing mechanism is that more actomyosin is activated by the presence of Mg++ @la). It has recently been suggested by Somlyo and Somlyo (25) that Mg may potentiate neurohypophyseal hormones by making available additional receptors for these peptides in the effector smooth muscle cells. Although the present study does not rule in or out this possibility, there are some indirect experiments in the recent literature that permit such a concept to be questioned. For example, Mg has been shown to potentiate, in addition to neurohypophyseal peptides and- certain amines (10, 11, 17a), barium-induced muscle (10 1 11) 1 contractions as well in aortic smooth barium being a vasoact ive substance that does not exert effects through a specific receptor. One would, therefore, be hard pressed to explain the potentiation of barium-
induced responses on the basis of magnesium making additional LLhormone receptors” available. A more likely explanation of Mg potentiation of barium contractions would be that Mg facilitates the translocation of a specific cellular Ca fraction necessary for the response to barium (10, 11). It must, however, be pointed out that the barium-induced contracti .ons are not associated with a shift in the doseresponse curve (EDso). Therefore, one must entertain the possibility that the two forms of potentiation (i.e., neurohypophyseal peptide and barium) may be mediated by different mechanisms. In the present study, an attempt has been made to determine whether the free amino group, OH group, aromatic group (i.e., phenylalanine), and basicity (and length of side chain) in positions 1, 2, 3, and 8, respectively, of the neurohypophyseal hormone structure are important in control of vasopressin potency by [Mg++10 in mammalian arterial muscle. Our findings indicate that the most basic peptide, i.e., [8-arginine]vasopressin, with arginine having a pk3 = 12.48, has its EDso with [Mg++J, shifted 6.8-fold to the left of that in the absence of [Mg++]., while [8-ornithinejvasopressin (with ornithine having a pka = 10.76) exhibits a much greater degree of potentiation in the presence of Mg, viz. lo-fold. These results tend to suggest that the length of the side chain in position 8 of the vasopressin molecule may be quite important in Mg potentiation in mammalian vascular muscle; i.e., ornithine has a shorter side chain than arginine* Our data with [8-lysine]vasopressin, with lysine having a pka = 10.28, but a side-chain length equivalent to that of arginine, could be used to support this tenet; the potentiation of lysine-vasopressin by Mg is approximately equivalent to that exhibited by
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MAGNESIUM,
NEUROHYPOPHYSEAL
HORMONES,
AND
VASCULAR
arginine-vasopressin, i.e., 6.9-fold (on the basis of ED&). In addition, these data suggest that although potentiation of neurohypophyseal peptides by Mg in mammalian vascular muscle may be somewhat dependent upon basicity in position 8 (e.g., [3-isoleucine, 8-leucine]vasopressin has a neutra1 molecule in position 8 and fails to exhibit potency shifts with Mg; i.e., the EDso is not shifted to lower conbasicity in this position centrations (Fig. I)), maximizing does not result in maximal contractile potency, e-g., compared to [8-ornithinelvasopressin. The data with l-Deamino-[8-argininelvasopressin indicate that 1.2 mM Mg shifts the ED 50 of this molecule almost ninefold; this is thus greater than the Mg potentiation that is seen with [8-argininelvasopressin. These findings tend to suggest that, at least with respect to rat aortic smooth muscle: a) the terminal, free amino group may not be important for Mg binding of the vasopressin hormone to its receptor; and b) the vasopressin molecule may actually interact (or bind more strongly) to Mg when a hydrogen is present rather than a free NH2 group. In addition, the present data suggest that although the phenolic group in position 2 is not critical for Mg-induced potentiation, the presence of an OH group in this position does enhance the interaction with Mg ions; e-g., although [Z-phenylalanine, 8-lysinelvasopressin and 1-Deamino-[Z-phenylalanine ,8-arginine] vasopressin are potentiated threefold each by [Mg++], (Fig. 2), [8-lysine] - and 1-Deamino-[8-argininelvasopressin are potentiated seven- and ninefold, respectively, by [Mg++], (Fig. 2) The findings with the substitution of isoleucine for phenylalanine in position 3 of [8-ornithine]vasopressin are of considerable interest. Interestingly, the EDso of the resultan t molecule, viz., [3-isoleucine, 8-ornithinelvasopressin is not changed by [Mg++]., quite unlike the tenfold potentiation by Mg of [8-ornithinelvasopressin (Fig. 2). These findings indicate that the interaction of Mg with an aromatic group (or phenylalanine) might be critical for potency of vasopressin contractions on mammalian vascuXar smooth muscle. Our findings concerning position 3 and Mg potentiation on vascular smooth muscle are thus quite diRerent from those reported for this position in uterine smooth muscle (30) It is rather interesting to note that of the three vasopressin analogues (or oxytocin derivatives) used here with isoleucine in position 3 (see Table 1 and Fig. 2), only one of them, viz., [2-phenylalanine , 3-isoleucine , &ornithine]vasopressin, has its ED 50 shifted by Mg (Fig. 2) ; and even here the displacement is only fourfold. These findings not only strengthen the notion that an aromatic group in position 3 of these hormones is important for Mg control of vasopressin potency (Le., to produce a shift in EDjo) on rat arterial smooth muscle (and maybe somatic vascular muscle in general), but could be used to suggest that there are two distinct, separate receptors for vasopressin and oxytocin in mammalian vascular smooth muscle. Several of the fmdings in this paper and in the literature could be utilized to buttress this tenet: I) oxytocin is an extremely weak contractile substance (e.g., exhibits highest ED& and lowest intrinsic activities or efficacies), not only on rat aortas described here, but on every other type of somatic microand macrovascular smooth muscle preparation so far l
1619
MUSCLE
examined findings).
in a variety of mammals (2, 5, 8, 25, and present 2) Although potency of oxytocin is not potentiated by 1.2 mM [MS++]. (i.e., shift the EDSO to lower concentrations) on rat aortic strips, both arginine-vasopressin and lysine-vasopressin ED 50’s are strongly potentiated by [Mg++],. This is thus different from that seen on rat uterine smooth muscle where vasopressin is only slightly more potentiated by Mg than is oxytocin (30). 3) On highly specialized vascular smooth muscle, such as that found in human umbilical arteries and veins, oxytocin exhibits not only a much greater afinity for its receptor site than does arginine-vasopressin but a much greater intrinsic activity as well (13) ; this is thus the reverse of that seen for these two posterior pituitary hormones on mammalian somatic blood vessels. 4) Arginine-vasopressin (preservative free) and its analogues have been shown to relax bovine coronary arteries (I), while oxytocin (preservative free) fails to induce similar action on these vessels (unpublished observations). 5) Low concentrations of synthetic oxytocin can induce potent relaxations of a variety of intact mesenteric microvessels in male rats, but vasopressin can only elicit contractile responses on these same vessels (4). Although these findings, collectively, do suggest that there may be separate receptor sites for oxytocin and vasopressin in mammalian vascular muscle, further work will be required before this hypothesis can be accepted. Specific competitive antagonists of vasopressin and oxytocin (17, 21) will be required to discern whether there are, indeed, two separate receptors for vasopressin and oxytocin in mammalian vascular muscle. The present study, in denronstrating that potentiation of nine structurally different neurohypophyseal hormones and analogues by external Mg ions is a direct function of the rat pressor potency of the molecules, indicates that Mgneurohypophyseal hormone interactions in vascular muscle may be somewhat different from those taking place in uterine smooth muscle. All the studies done so far with uterine smooth muscle indicate that potentiation of neurohypophyseal peptides by Mg++ is inversely related to the uterotonic potency of these molecules, not directly (14, 16, 18, 20, 25, 30, 3 1). Th is is probably due to intrinsic differences between the uterine smooth muscle and vascular muscle receptors for these posterior pituitary hormones. Such fundamental differences could prove useful in the characterization of the receptors for these hormones in blood vessels. I am grateful for the excellent technical assistance provided in these studies by Marjorie K. Nicodemus and Charles F. Reich. I am indebted to the late Dr. R. Bircher and Dr. B. Berde of Sandoz Ltd., as well as to Dr. R. Walter, Mt. Sinai School of Medicine, for generously supplying the synthetic peptides used in these studies. This study was supported in part by Public Health Service Research Grant HE-18015 and Grant HL-12462 from the National Heart and Lung Institute. Part of this study was performed during the tenure of B, M. Altura of a Research Career Development Award from the National Institutes of Health, Public Health Service. Requests for reprints should be sent to: B. M. Altura, Dept. of Physiology, Box 31, State University of New York, Downstate Medical Center, Brooklyn, N. Y. 11203. Received
for publication
14 June
1974.
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B.
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M.
ALTURA
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