Antagonism of Responses to Arginine Vasotocin by Its Structural Analogs in the Bullfrog, Rana catesbeiana’ MINORU UCHIYAMAAND~ETER Department
of Oral Japan;
Physiology, and Department
School of Dentistry at Niigata, Nippon of Physiology, Faculty of Medicine, Edmonton, Alberta, Canada
PANG Dental University
University, Niigata of Alberta,
Accepted December 14, 1989 Antagonism of arginine vasotocin (AVT) actions in vivo by synthetic AVT analogs was studied in bullfrogs. In addition, one analog was examined for its in vitro effect on water flux in the urinary bladder and on vascular contraction in a dorsal aortic preparation. AVT and its analogs were injected into conscious bullfrogs while blood pressure and urine flow rate were recorded simultaneously. d(CH,),Tyr(Me)AVT induced a slight, but not statistically significant, antidiuresis and a clear vasopressor response. d(CH,),Tyr(Et)OVT and d(Et,)Tyr(Et)OVT did not show any intrinsic effects. These analogs partially blocked the antidiuretic effects of AVT and completely blocked the pressor effects of AVT. In studies in vitro, 1 p,M d(Et,)Tyr(Et)OVT antagonized AVT-stimulated water flux, whereas 10 nM competitively inhibited (by about 50%) the vasocontraction induced by AVT. These results suggest that d(Et,)Tyr(Et)OVT has no intrinsic activity but retains a relatively high receptor affinity, thereby producing effective antagonism of AVT in target cells of vascular smooth muscle. On the other hand, this antagonist showed no detectable intrinsic activity and appeared to be a weaker antagonist in target cells of the urinary bladder. This suggests that there might be two types of AVT receptors in bullfrogs. o 1%~ Academic press, I~C.
It is known that arginine vasotocin (AVT) is the antidiuretic and pressor substance present in the neurohypophysis of nonmammalian vertebrates, whereas most mammals possess arginine vasopressin (AVP). In the case of AVP, two types of AVP receptor can be distinguished: Ca’+-dependent V, receptors found in the smooth muscles and hepatocytes, and CAMP-dependent V, receptors found in the kidney (Michell et al., 1979). On the other hand, the presence of two types of AVT receptor is unknown, although AVT has been considered the most primitive of the active neurohypophysial hormones and may be ancestral to all other neurohy’ Part of this paper was presented at the First Congress of the Asia and Oceania Society for Comparative Endocrinology (AOSCE). Nagoya, Japan, Nov. 4-7, 1987.
pophysial hormones (Sawyer and Pang, 1977). In some nonmammalian tetrapods, AVT can cause both vasopressor and tubular antidiuretic responses, suggesting that there are both tubular and vascular receptors, although it does not mean that they differ (Chan, 1977; Pang, 1977). The presence of AVP receptors that differ pharmacologically in mammals also suggests this possibility. In structure-activity studies of AVP, the discovery of analogs that block the antidiuretic and/or vasopressor effects in viva and in vitro has been important (Sawyer and Manning, 1984). AVP analogs were used to investigate the characteristics of the hydroosmotic receptors in toad urinary bladder (Fahrenholz et al., 1986; Mann et af., 1986). Mann et al. (1986) suggested that there are important differences between amphibian and mammalian antidiuretic hor355 0016-6480190 $1.50 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
mone receptors with regard to structureactivity relationships. Similarly, several studies have utilized analogs of AVT (Jard and Morel, 1963; Morel and Jard, 1963; Berde and Boissonnas, 1968; Eggena et al., 1968: Fahrenholz et al.. 19861. In anurans. however, these studies have been confined to the antidiuretic and the hydroosmotic actions, and little has been done with vascular actions. The nresent studies were undertaken to determine whether new synthetic analogs would antagonize the in vivo actions of AVT in bullfrogs. In addition, an analog was used in vitro to investigate the possibility of two types of AVT receptor in bullfrogs. MATERIALS
Bullfrogs, Rana catesbeiana, were purchased from a dealer in Sango City, Saitama Prefecture. and maintained in running tap water in the laboratory. In Go experiments. Bullfrogs were anesthetized by immersion in 0.1% tricaine methanesulfonate (MS 222, Sankyo). Both ureters were cannulated with PE-50 polyethylene tubing by the dorsal approach described by Uranga and Sawyer (1960). The right iliac artery and the right musculocutaneous vein were also cannulated for blood pressure recording and intravenous injection, respectively. After the operation the animals were kept in water in opaque plastic boxes overnight or longer before use in experiments. Ureteral urine was allowed to flow freely and was monitored with a drop counter (Model lA, Naito). The drop volume was determined before, and rechecked at the completion of, each experiment. In the first experiment, urine flow rate and blood pressure were monitored for an initial (90 min) control period in conscious frogs, and then AVT (200 &kg) or an analog (10 kg/kg) was injected intravenously. Changes in urine flow rate and blood pressure were measured during the following half-hour period. In a second set of experiments, urine flow rate and blood pressure were monitored for an initial (90 min) control period to establish basal levels, and then AVT (200 rig/kg) was injected intravenously to measure a normal full antidiuretic and vascular response. At the end of antidiuresis (as judged by a return of the urine flow rate to its control level), an analog (IO &kg) was injected. Five minutes after injection of the analog, a challenge dose of AVT (200 rig/kg) was administered. and changes in urine flow rate and blood pressure followed for two consecutive half-hour periods.
AND PANG Measurement
to A VT.
Frogs were doubly pithed, and their urinary bladders were resected and tied to the ends of glass cannulas with the mucosa on the inside and the serosa on the outside of the bladder sac. Bladders were filled with dilute Ringer’s solution (1:5 dilution) and suspended in full-strength Ringer’s solution of the following composition, in mM: NaCl, 110; KCI, 3.5; CaC&. 1; MgCl, I ; dextrose, 5.5: Tris(hydroxymethyl)aminomethane HCI, 10. The pH of this solution was adjusted to 7.4. and its osmolality was 235 mOsm/kg H,O. as determined with an osmometer (Yanako, Inc.). These preparations were preincubated for two 30-min periods in a fresh bath of Ringer’s solution bubbled with room air. AVT and an analog were added to the serosal side. Net osmotic water flux across the bladder wall was measured gravimetrically according to the method of Bentley (Bentley, 1958). Briefly, contralateral hemibladders were each exposed simultaneously to two different solutions (with or without hormone or with different concentrations of hormone) for 30 min. Water flux was measured as the decrease in sac weight at the end of each incubation neriod. Sacs were blotted gently on gauze before each weighing. Both mucosal and serosal solutions were changed after each weighing, and then the bladders were incubated for two 30min periods in a fresh Ringer’s solution to wash out drugs. After the flux recovered to its control level, bladders were incubated with increasing concentrations of AVT for 30 min. Concentration-response relationship was developed for AVT. Inhibition of AVTstimulated flux was determined by incubations of paired bladders in the absence or presence of the analog with a fixed concentration of AVT (100 PM). Measurement
Frogs were doubly pithed, and the dorsal aortae were removed and placed in oxygenated Ringer’s solution. After removing the surrounding tissues under a dissetting microscope, the aortae were cut into helical strips. Each aortic strip was then arranged in a tissue holder assembly and placed in a tissue chamber containing 10 ml Ringer solution, as mentioned previously in the urinary bladder experiment. The bath solution was continuously aerated with 95% Oz and 5% COz and maintained at 21-23”. The pH of this solution was kept between 7.0-7.4 during the experiment. The free end of the aorta was attached to a force-displacement transducer and the tension was recorded (Nihon Kohden, a developed version of Model SB-IT, and a Nihon Kohden multipurpose polygraph). Tissues were allowed to equilibrate for 1 hr under a resting tension of 400 mg. After the equilibration period, AVT and an analog were examined. Inhibition by the analog was tested by administration of AVT IO min after the analog treatment. The contractile effect of AVT and the inhibitory effect on the AVT-induced response produced by the analog were expressed as the change
8. Typical parathyroid gland. Bar shows 50 w. 9. Enlarged photograph of typical parathyi roid gland, showing the whorled arrangement of parenchymal cells. Note the difference in size of the epithelial cells between the central portion and the periphery. Bar shows 50 pm. HE staining. FIG. FIG.
of the crab-eating frog was 3.40 m,Wliter. Although there are no data about the serum Mg level in freshwater frogs, the average value (1.30 mM/liter) for the crab-eating frog was slightly higher than the level (about 1.1 mM/liter) for the common newt Cynups pyrrhogaster (Oguro and Uchiyama, 1975). These facts show that in crabeating frogs the levels of serum divalent salts are certainly higher than those in freshwater amphibians. The ultimobranchial gland of the crabeating frog is composed of a single follicle or multiple follicles. This is fundamentally similar to the feature reported in freshwater anurans (see, Robertson, 1971). However, some minor differences were recognized. For example, in R. pipiens, the parenchy-
ma1 surface of the gland is sometimes complicatedly rugged (Robertson, 1988). In the crab-eating frog, however, the surface of the parenchyma was smooth even in the invaginated gland. This may be due to the difference in manner of growth of the gland. In the crab-eating frog, the follicle wall seems to grow to the inside of the gland, but in R. pipiens it grows to the outside. Furthermore, in the invaginated glands of the crab-eating frog, blood vessels were found inside the parenchyma. These vessels may originate from those located in the periphery of the gland, which entered the interior with invagination of the follicle wall. This may be related to the secretion of hormone into the blood vessels, as mentioned below. The calcitonin immunoreaction of the ul-
AVT 11.89 9.56 12.45 10.88 12.58 14.65
2 + + k 2 i
1.27 2.04 2.09 3.11 0.85 0.87
2.39 3.28 5.75 3.05 5.10 2.51
k t k ? t 2
0.62” 0.67’ 1.25’ 0.56’ o.43b 0.70”
later AVT was tested intravenously.
14 8 12 9 3 3 f ? k t 2 ?I
1.64’ 1.26 I .80’ 1.56 0.58” 0.51”
29.2 33.6 32.4 29.1 32.3 34.2 Values are means 2 SE;
4.90 3.73 6.10 6.64 3.08 1.27
Blood pressure change (mm Hg) ~__ -t 0.9 5.1 2 0.3” 2 2.9 0. I 2 0.4d 2 1.8 -0.4 i 0.4” r 0.9 4.9 k 0.8” -+ 1.7 4.9 2 0.9 -+ 3.5 5.1 k 0.8 __-,I. number of frogs used.
TABLE 2 OF ANALOGS ON URINE FLOW RATE AND BLOOD PRESSUREIN BULLFROGS -- ___ _I_. ~ __. Mean resting Urine flow rate (ml/kg-hr) __~___ -__ -__ Dose blood pressure 0.5 hr Initial I hr (r&kg) (mm Hg) -. - n .-____
AVT alone 200 IO AVT 200 d(CHJ,Tyr(Et)OVT d(EtJTyr(Et)OVT 10 AVT 200 d(CH,),Tyr(Me)TOT 10 AVT 200 d(CH,),Tyr(Me)AVT 10 AVT 200 d(CH,),Tyr(Me)VDAVP 10 AVT 200 - ~~ ___. .Nor?. Each frog was injected with an analog, and 5 mitt ” P < 0.001 compared with initial value in each group. ” P < 0.01 compared with initial value in each group. ’ P < 0.05 compared with initial value in each group. ‘P < 0.001 compared with AVT-treated control.
5 a V i2 0
C 2 7
AVT AND ANALOGS
increase of water permeability (Fig. I). The minimum effective concentration of AVT was less than 10 pM and the maximum response was obtained with 1 nM AVT. The concentration of AVT producing a halfmaximal response (ED,,) was 37.9 pM. By contrast, d(Et,)Tyr(Et)OVT at concentrations from 1 to 100 nM did not exhibit any intrinsic action on water movement. A high concentration (1 PM) of d(Et,)Tyr(Et)OVT significantly inhibited the water movement normally induced by AVT (100 PM). Measurement to AVT
of Aortic Strip Responses
AVT contracted the aortic strips in a concentration-dependent manner (Fig. 2). The minimum effective concentration of AVT was 0.1 nM. The ED,, value of AVT was 3 1.2 nM. d(Et,)Tyr(Et)OVT (1 IN to 1 PM)
did not contract the aortic strips. The response of AVT-induced contraction was shifted to the right in the presence of d(Et,)Tyr(Et)OVT (10 nM). The dose of this analog (10 nM) inhibited the vasocontraction induced by AVT by about 50%. Lineweaver-Burk plots (Fig. 3) of AVT alone and AVT plus different concentrations of d(Et,)Tyr(Et)OVT met at the I/ effect axis (the inverse of tension). In the response to AVT, there was a high correlation between the in vitro and in vivo studies. d(Et,)Tyr(Et)OVT inhibited competitively the AVT-evoked vasoconstriction. The inhibitory potency of d(Et,)Tyr(Et)OVT on AVT-induced water flow was low. DISCUSSION
The present studies have reconfirmed previous observations that exogenous AVT induces distinct antidiuretic and vasopresI -
+* -+----+ 10 11 AVT and analog,
FIG. 1. Concentration-response curve for arginine vasotocin (AVT) and effects of d(Et,)Tyr(Et)OVT on AVT-stimulated water flux in frog urinary bladder. Solid circles, AVT; open circles, analog; solid square, analog (10 nM) + AVT; solid triangle. analog (100 n&f) + AVT; solid star, analog (1 p&Z) + AVT. Abscissa, -log of AVT and analog concentration; ordinate, increase (A) in water flux: Cont., control. Points represent means 4 SE, n = 6. *P < 0.01 compared with AVT-treated control.
9 8 AVT and analog,
FIG. 2. Concentration-response curve for action of AVT and d(Et,)Tyr(Et)OVT on aortic strips. Solid circles, AVT; open circles, analog: solid triangles, analog (10 IN) + AVT. Abscissa, -log of AVT and analog concentration; Ordinate, increase (A) in tension. Points represent means k SE, n = 6 to 10.
FIG. 3. Lineweaver-Burk plot between reciprocal of AVT concentration and that of tension. Solid circles, AVT; solid squares, d(Et2)Tyr(Et)OVT (1 nM) + AVT: solid triangles, d(Etz)Tyr(Et)OVT (IO nM) + AVT.
SOT responses in bullfrogs (Pang et al., 1980; Uchiyama and Pang, 1985). In the present in vivo experiment, the agonistic or antagonistic potencies of same analogs of AVT were compared. The analogs used in the present studies possess large alkyl substituents on the B carbon at position 1 and (O-methyl)- or (O-ethyl)-tyrosine at position 2, which are important for antagonism of the vasopressor and the antidiuretic effects of AVP in rats (Sawyer et al., 1981a). However, these analogs differ in the 4 (Gln, Thr, Val) and 8 (Arg, Leu, Orn) positions. Large doses of these artificial peptides induced different pharmacological effects on blood pressure and urine flow rate in bullfrogs. Berde and Boissonnas (1968) reported that the structural requirements for the amino acid at position 8 are more limiting at the vasopressor receptor site than at the antidiuretic site in rats. Pang and Sawyer (1978) observed that mesotocin induced vasodepressor responses in bullfrogs. Stiffler et al. (1984), working with larvae of Ambystoma tigrinum, suggested that the amino acid at position 8 may be essentia1 for the diuretic action of mesotocin through an increase in the glomerular filtration rate.
On the other hand, AVT is an antidiuretic and pressor substance. In the present study, the Arg’ analog, d(CH,),Tyr(Me)AVT, showed vasopressor agonist and induced slight, but not statistically significant, antidiuresis. When arginine was replaced by ornithine at position 8, the agonistic actions of the analog disappeared and antagonistic effects of the analog for AVT were emphasized. These findings show that the amino acid at position 8 of AVT may participate in the characteristics of a vascular receptor of AVT. The data presented in Table 2 show that d(EtJTyr(Et)OVT is a potent antagonist of AVT in bullfrogs. This analog blocked the vasopressor effect of AVT, but its anti-antidiuretic activity was relatively low. In rats, this analog had very low antidiuretic activity and potent antivasopressor and antioxytocic activities (Bankowski et al., 1980). The characteristics of the vascular receptor for AVT may, therefore, be similar to those of the V, receptor for AVP. In our in vitro studies, it was observed that the hydroosmotic response to AVT in the urinary bladder is more sensitive than the vasopressor response to AVT in vascular smooth muscle. This is consistent with
AVT AND ANALOGS
in viva experiments showing that much higher doses of exogenous AVT are required for the pressor response than for the antidiuretic response in bullfrogs and Japanese toads (Pang et al., 1980; Uchiyama, unpublished data). Our data show that d(Et,)Tyr(Et)OVT had little intrinsic activity and appeared to be a weak antagonist in target cells in the urinary bladder. However, this analog is a potent antagonist of pressor activity and vascular smooth muscle contraction (Table 2; Fig. 2). This observation is consistent with the hypothesis that the AVT receptors of vascular smooth muscle may be different from the AVT receptors in the urinary bladder of bullfrogs. In the in viva study, this analog partially blocked the antidiuretic effect of exogenous AVT. It is considered that the vascular antagonist blocked the decrease in glomerular filtration rate, but did not block the change in water permeability of the renal tubules. This is consistent with previous results for renal functions such as glomerular filtration rate and free water clearance in bullfrogs and Japanese toads (Uchiyama et al., 1987; Uchiyama, unpublished data). We have suggested previously that the renal and vascular responses to AVT are related and that there are four basic patterns of response which can explain diuresis and antidiuresis in fish and tetrapods, respectively (Pang et al., 1982). This analog, therefore, is expected to be a valuable tool as a vasopressor antagonist in a variety of studies on the presumptive physiological roles of AVT. ACKNOWLEDGMENTS The authors thank Dr. W. H. Sawyer at Columbia University and Dr. M. Manning at Medical College of Ohio for supplying the vasotocin analogs.
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IN BULLFROG potent in vivo
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