ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 212, No. 2, December, pp. 660-66’7, 1981

Modulation of Parathyroid Hormone-Sensitive Adenylate Cyclase and Arginine Vasopressin-Sensitive Adenylate Cyclase by Calcium and GTP

TAI C. CHEN AND JULES B. PUSCHETT

Department sf Medicine, Rend-Ekctrolyte Division, University of Pittsburgh Schmol of Medicine, Pittsburgh, Pennsylvania 15261

Received January 26, 1981, and in revised form July 10, 1981

Arginine vasopressin (AVP)- and parathyroid hormone (PTH)-sensitive adenylate cy- clase were studied in the renal tissue of thyroparathyroidectomized dogs. The results indicate that AVP-sensitive adenylate cyclase activity was highest in the inner medulla followed by the middle medulla, outer medulla, and cortex, in declining order. In contrast, PTH-sensitive adenylate cyclase was absent in the inner medulla, and the highest stim- ulation was found in the cortex with lesser activity in outer and middle medulla. When 1 mM EGTA was included in the incubation mixture, the addition of both AVP and PTH to the middle medullary homogenate resulted in additive responses suggesting two sep- arate receptors for each hormone. This EGTA-induced additive effect was eliminated by the addition of calcium into the system, indicating that calcium concentration may be critical in modulating the interaction of AVP- and PTH-sensitive adenylate cyclase. In contrast to some previous reports, a particulate fraction prepared from the middle med- ullary tissue was completely insensitive to either AVP or PTH. Hormonal sensitivity was restored by the addition of GTP or the supernatant.

There is considerable evidence that the physiologic actions of parathyroid hor- mone (PTH)’ and vasopressin (VP) in the kidney are mediated through the stimu- lation of membrane-bound adenylate cy- clase (l-11). More recently, characteriza- tion of the receptors by radiolabeled hor- mones and their analogs has established the correlation between binding proper- ties and adenylate cyclase stimulation (12 15). Since a hormone receptor is a distinct entity, separate from adenylate cyclase (16), an understanding of the intermediate mechanisms involved in the receptor-me- diated activation of the enzyme is of great importance. Rodbell and his associates have identified four allosterically linked effector sites from their study of the glu- cagon-sensitive adenylate cyclase system in rat liver membrane (17). Occupation of

1 Abbreviations used: PTH, parathyroid hormone; VP, vasopressin; bPTH-(l-34), bovine parathyroid hormone fragment l-34, AVP, 8-arginine vasopres- sin; TPTX, thyroparathyroidectomized.

three of these (by glucagon, GTP, and di- valent cations) enhances activity, whereas occupation of the adenosine site results in an inhibition of enzyme activity. Further- more, Farfel et al. have recently reported a defect in receptor-cyclase coupling pro- tein activity which could contribute to the PTH insensitivity of patients with pseu- dohypoparathyroidism (18).

The importance of calcium in the hor- mone-sensitive adenylate cyclase system has been suggested in a number of systems (19,20). The responses of both adipose and adrenal cortex adenylate cyclases to ACTH have been shown to be dependent on Ca2+, although Ca2’ at concentrations above 10e3 M causes inhibition. Evidence has been presented that Ca2+ is not required for binding of ACTH to the receptor in the adrenal cortical system (21), whereas the binding of labeled PTH to bovine renal cortical membranes has been shown to be modulated by calcium ions (22). Given the importance of calcium concentration and GTP in a number of other adenylate cy-

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660

EFFECT OF Ca and GTP ON CANINE RENAL ADENYLATE CYCLASE 661

clase systems, and the existence of little if any data with regard to their effects on the interaction between AVP- and PTH- sensitive adenylate cyclase, we studied the following aspect of this interaction: (i) the AVP- and PTH-sensitive adenylate cy- clase in cortical tissue as well as three portions of the medullary tissue of dog kidneys; (ii) modulation of the two hor- mone-sensitive adenylate cyclase systems and their interaction with calcium and EGTA; and (iii) the effect of GTP on the two hormone-sensitive adenylate cyclase systems in canine renal tissue.

MATERIALS AND METHODS

Radiolabeled cyclic [‘HIAMP (specific activity 40 Ci/mmol) was obtained from New England Nuclear Corporation (Boston, Mass.). ATP (cat. No. A 2383) and GTP (cat. No. G 5756) were purchased from Sigma Chemical Company (St. Louis, MO.). bPTH-(l- 34) (lot No. 1220, 6060 U/mg) was obtained from Beckman Instruments Inc. (Palo Alto, Calif.); syn- thetic 8-arginine vasopressin (AVP) (lot No. Y-2316, 300 U/mg) from Schwa&Mann (Orangeburg, N. Y.). All other chemicals were reagent grade and were obtained from commercial sources.

Normal hydropenic mongrel female dogs were thy- roparathyroidectomized (TPTX) at least 2 days be- fore the experiments. The kidneys from thyropara- thyroidectomized dogs were removed rapidly and placed in ice-cold saline. The cortex and three regions of the medulla (outer, middle, and inner zones) were dissected according to the anatomical description of Trump and Bulger (23). After weighing, the cortex was homogenized in 50 vol (w/v) of 2 mM Tris-HCl buffer, pH 7.4, in a Potter-Elvehjem homogenizer fitted with a Teflon pestle. Medulla from each region was homogenized in a Polytron homogenizer, Model PT 10-35, fitted with a PTlOST generator (Brink- mann Instruments, Westbury, N. Y.).

For the preparation of a particulate fraction, tissue was homogenized in 50 vol (w/v) buffer containing 2 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The ho- mogenate was centrifuged at 25,300g for 15 min; the supernatant fraction was removed and the pellet ho- mogenized again in 50 vol of the homogenizing buffer and centrifuged. The second pellet obtained was again resuspended and centrifuged. The final pellet was resuspended in 25 vol of 2 mM Tris-HCl buffer, pH 7.4. This final suspension of washed particulate material was designated Pa. The protein concentra- tion of these preparations ranged from 1.8 to 2.5 mg/ml.

Measurement of adenylate cyclase activity was

carried out as previously described (24). Cyclic AMP was measured by Brown’s binding assay (25). Protein was determined according to the method of Lowry et aL (26).

RESULTS

The effect of various concentrations of synthetic Sarginine vasopressin (AVP) on the adenylate cyclase activity of homoge- nates of the inner medulla from dog kid- ney is shown in Fig. 1. There is no signif- icant difference in percentage stimulation over basal activity in the presence or ab- sence of 1 mM EGTA. However, both basal and hormone-stimulated enzyme activity were markedly increased in the presence of EGTA. One-half maximal stimulation in enzyme activity was achieved with about 1 nM AVP in the presence of EGTA. These data for AVP-sensitive adenylate cyclase in the inner medulla (Fig. 1) are compared to that for AVP in middle me- dulla in Fig. 2B. Adenylate cyclase was less responsive to AVP in middle medulla than in inner medulla in a medium con- taining 1 mM EGTA. However, there was essentially no difference in the concentra- tion of hormone which caused one-half maximal stimulation in enzyme activity. Figure 2A shows the effect of graded con- centrations of synthetic bovine PTH-(l-

FIG. 1. Dose-response curves of adenylate cyclase responsiveness to garginine vasopressin in homog- enates of dog renal inner medulla in the absence (closed circles) or presence (open circles) of 1 mM EGTA. Each point is the mean + SEM of five de- terminations from a typical experiment.

CHEN AND PUSCHETT

PTH [llrnl 8 - arginine vasopressin (NM)

FIG. 2. Dose-response curves of the adenylate cyclase response to hPTH-(1-34) and 8-arginine vasopressin in dog renal middle medullary homogenates in the presence of 1 mM EGTA. Each point is the mean + SEM of five determinations from a single experiment.

34) on adenylate cyclase activity of middle PTH has activity. Only in the inner me- medulla. Maximal response was reached dulla was PTH without effect. Further- with a concentration of 0.1 PM of the hor- more, the addition of 1 mM EGTA in- mone, which reached levels of 50 pmol creased both the basal and hormone- cAMP/mg protein/min. By contrast, a stimulated adenylate cyclase activities. maximal stimulating effect of 42.5 pmol The effect of EGTA on AVP- and PTH- cAMP/mg protein/min was reached with sensitive adenylate cyclase was further 0.003 PM AVP. studied in homogenates of middle medulla

Next, AVP- and PTH-sensitive adenyl- since in this region both hormones showed ate cyclase were studied in cortical and activity. As shown in Table II, in the ab- outer, middle, and inner medullary tissue. sence of EGTA the combined effects of the Maximal doses of synthetic bPTH-(1-34) two hormones at their maximal doses did (0.4 PM ) and synthetic AVP (0.28 I.LM ) were not exceed the response produced by PTH employed. As shown in Table I, AVP-sen- alone. However, an additive effect was ob- sitive adenylate cyclase was found in ho- served when 1 mM EGTA was present in mogenates from all four regions. The ac- the incubation mixture. A similar inter- tivity was the highest in the inner medulla action of these two hormones was also in either the presence or absence of 1 mM found in the renal cortex (Table III). That EGTA (about a twofold stimulation). Fur- the EGTA-induced additive effect of these thermore, AVP was least stimulatory in two hormones could be eliminated by add- the cortex, especially when EGTA was not ing calcium ions into the system contain- added. PTH-sensitive adenylate cyclase ing EGTA is shown in Table IV. Addi- was absent in the inner medulla, while the tionally, it should be noted that high highest stimulation was found in the cor- calcium concentrations (1 InM) inhibited tex and outer medulla. PTH-stimulated both the basal and AVP-stimulated ade- CAMP formation was higher than that in- nylate cyclase activities. duced by AVP in all three regions in which The properties of the hormone-sensitive

EFFECT. OF Ca and GTP ON CANINE RENAL ADENYLATE CYCLASE 663

TABLE I

THE DISTRIBUTION OF AVP AND PTH-SENSITIVE ADENYLATE CYCLASE IN VARIOUS REGIONS

OF DOG KIDNEY

Homog- enate from Addition”

Adenylate cyclase activityb (pm01 CAMP/

mg protein/min)

+1 InY -EGTA EGTA

Cortex None 10.8 f 0.6 15.8 + 0.6 bPTH-(1-34) 43.0 * 3.0’ 66.0 k 0.8* AVP 13.6 + 0.8; 21.6 + 0.7*

Outer None 9.8 + 0.6 20.4 + 2.4 medulla bPTH-(1-34) 44.6 + 3.3’ 68.6 + 3.2’

AVP 16.1 ? 0.9* 28.7 f 1.3’

Middle None 18.7 k 0.9 30.9 5 1.8 medulla bPTH-(l-34) 32.7 f 0.6* 50.6 f 1.5*

AVP 24.5 * 1.2* 44.2 + 2.08

Inner None 20.2 f 1.1 30.2 + 1.5 medulla bPTH-(l-34) 21.0 + 1.6 29.2 + 1.0

AVP 40.5 k 3.0’ 69.2 k 2.1’

a Maximal doses of bPTH-(l-34) (0.4 PM) and AVP (0.23 PM) were employed in the incubation.

b Mean + SEM for five replicate samples in a single ex- periment.

* P <: 0.01, comparing control observations with hormone response.

adenylate cyclase were further studied in the membrane preparation. Following centrifugation and repeated washing of the particulate material (Pa) from middle medulla, the stimulatory effect of AVP (0.28 PM) was no longer evident (Fig. 3). However, at this concentration, the hor- mone had been demonstrated to have a stimulatory effect in whole homogenates (Fig. 2B). The addition of 10 PM GTP (final concentration) to the P3 fraction partially restored the ability of AVP to stimulate adenylate cyclase (Fig. 3). In the absence of GTP, 1 mM EGTA alone had no effect on hormone stimulation. Only the addition of both 10 PM GTP and 1 mM EGTA to the P3 fraction restored AVP stimulation to the enzyme. Although the percentage stimulation over basal activity was re- stored to the same degree as in the original homogenate, the absolute stimulation (pmol CAMP formed/mg protein/min) of the reconstituted system was significantly less than that in the whole homogenate.

The effects of PTH on the adenylate cy-

clase activity of this particulate (Ps) frac- tion in the presence and absence of GTP and EGTA are shown in Fig. 4. The PTH- sensitive adenylate cyclase from the same (P3) preparation obtained in middle me- dulla required only GTP for its coupling activity, although EGTA indeed increased both basal and hormone-stimulated activ- ities (Fig. 4).

DISCUSSION

It is generally accepted that PTH and vasopressin initiate some of their actions on target cells by interacting with specific receptors resulting in the stimulation of adenylate cyclase. Our study of the dis- tribution of AVP-sensitive adenylate cy- clase in dog kidney shows that it is not confined to renal medulla (Table I). These results are in agreement with those re- cently reported by Imbert et aL (lo), Dousa (7), and Charbardes et al. (9, 11). In ex- periments performed in isolated renal tu- bules from rabbits (10) and human kid- neys (ll), the authors showed the existence

TABLE II

MODULATION OF PTH-SENSITIVE ADENYLATE

CYCLASE AND AVP-SENSITIVE ADENYLATE CYCLASE INTERACTION IN HOMOGENATES OF DOG RENAL

MIDDLE MEDULLA BY EGTA

Adenylate cyclase activityb (pmol cAMP/mg

protein/min)

Addition” +l mM

-EGTA EGTA

None 19.7 f 1.4 32.2 -e 2.6

bPTH-(1-34) 36.0 f 2.1* 54.9 It 1.8** AVP 26.4 + 0.8* 47.8 -c 1.8*

bPTH-(l-34) and AVP 35.0 f 1.6* 69.6 It 1.9**

’ Maximal doses of bPTH-(1-34) (0.4 PM) and AVP (0.28 MM) were employed in the incubation.

” Mean f SEM for five replicate samples in a single experiment.

* P < 0.01, comparing control observations with hormone response.

** P value for comparison of hPTH-(1-34) alone vs bPTH-(1-34) plus AVP in the presence of 1 mM EGTA is cO.01.

664 CHEN AND PUSCHETT

TABLE III MODULATION OF PTH-SENSITIVE ADENYLATJZ

CYCLASE AND AVP-SENSITIVE ADEN~ATE CYCLASE INTERACTION IN HOMOGENATES OF DOG RENAL

CORTEX BY EGTA

Adenylate cyclase activityb (pmol cAMP/mg

protein/min)

Addition” +l mM

-EGTA EGTA

None 10.5 + 0.4 11.4 + 0.6 bPTH-(1-34) 36.8 f 2.0* 61.0 k 1.6** AVP 15.7 + 0.6* 22.4 + 0.8* bPTH-(l-34) and AVP 36.6 f 0.9* 71.3 + 2.2**

’ Maximal doses of bPTH-(1-34) (0.4 PM) and AVP (0.28 PM) were employed in the incubation.

’ Mean + SEM for five replicate samples in a single experiment.

*P < 0.01, comparing control observations with hormone response.

** P value for comparison of bPTH-(1-34) alone vs bPTH-(1-34) plus AVP in the presence of 1 mM EGTA is cO.01.

of AVP-sensitive adenylate cyclase in the late portions of the distal convoluted tu- bule and in cortical and medullary seg- ments of the collecting tubule. The absence of PTH-sensitive adenylate cyclase in the inner medulla and the presence of the en-

zyme in the middle medulla, outer me- dulla, and cortical portions of the kidney are also in agreement with data obtained from isolated human renal tubules (11). In the latter studies, PTH (10 IU/ml) in- creased adenylate cyclase activity in the convoluted and straight proximal tubule, in the medullary and cortical portions of the thick ascending limb, and in the early portion of the distal convoluted tubule. No PTH-sensitive adenylate cyclase was found in medullary collecting tubule.

Although adenylate cyclase was less re- sponsive to AVP in middle medulla than in inner medulla (Fig. 1 and Fig. 2B), there was no difference in the concentration of hormone which caused one-half maximal stimulation in enzyme activity. These data suggest that the AVP-sensitive adenylate cyclase in these two regions of dog kidney may be identical. The K, for hormone stimulation is in agreement with that re- ported by Charbardes et al. for collecting tubules of human nephrons (11). This con- centration is substantially lower than that found for S-lysine vasopressin-stimulated porcine renal plasma membrane (27). However, the variation in concentration may well be due to species difference (28). The additive nature of the response to the hormones (Table II) suggest that, in the presence of 1 mM EGTA, two different

TABLE IV

EFFECTS OF EGTA AND OF CALCIUM ADDITION TO THE EGTA-CONTAINING MEDIUM ON THE ABILITY OF bPTH-(l-34) AND AVP TO STIMULATE ADENYLATE CYCLASE OF DOG RENAL MIDDLE MEDULLA

Adenylate cyclase activityb (pmol cAMP/mg protein/min)

Addition” No EGTA +l mM EGTA

+l mM EGTA

+O.l mM Ca”

+l mM EGTA +l mM CA”

None 20.2 + 1.1 23.8 2 0.3 25.3 4 0.8 9.6 + 0.5 bPTH-(1-34) 40.7 + 1.6* 55 + 1.6** 58.3 2 2.08 23.9 + 0.7* AVP 25.5 f 1.9* 31.5 + 1.7 31.9 k 1.7* 12.5 + 0.8’ bPTH-(l-34) and AVP 42.3 + 1.7* 61.5 + O.l** 59.1 zk 2.8* 24.4 + 0.5*

” Maximal doses of bPTH-(1-34) (0.4 PM) and AVP (0.28 PM) were employed in the incubation. * Mean f SEM for five replicate samples in a single experiment. *P c 0.01, comparing control observations with hormone response. ** P value for comparison of bPTH-(1-34) alone vs bPTH-(1-34) plus AVP in the presence of 1 m?d EGTA

is 10.01.

EFFECT OF Ca and GTP ON CANINE RENAL ADENYLATE CYCLASE 665

1

FIG. 3. Effects of EGTA and GTP on the adenylate cyclase response of a particular membrane fraction (Pa) obtained from dog renal middle medulla to AVP. The data represent the means + SEM for five rep- licate samples in a single experiment. The concen- tration of AVP employed was 0.28 PM.

hormone receptors were involved in the stimulation of adenylate cyclase by PTH and/or AVP.

Guanyl nucleotides are endogenous components of the cell cytosol, which have been demonstrated to be required for the “coupling” of adenylate cyclase to dopa- mine (29) and P-adrenergic receptors (30, 31). Furthermore, these nucleotides have been shown to enhance the stimulation of adenylate cyclase by various hormones (17). In this report, we present evidence that GTP is essential for the stimulation of adenylate cyclase in middle medulla by PTH and AVP. In contrast to our results, Herman et al. (32) have reported the ap- parent lack of a GTP requirement for AVP activity in rat kidney tissue. This discrep- ancy may either be due to species differ- ence, or perhaps to differences in the methodology in the preparation of the membrane fraction. The ability of GTP to restore hormone sensitivity (Figs. 3, 4) strongly suggest that GTP may be the en- dogenous coupling factor for PTH and AVP in dog kidney as is the case in rat brain caudate nucleus (29), rabbit cere-

bellum (30), and in a human astrocytoma cell line 132-1Nl (31).

Some reports have suggested that a minimal amount of calcium is required for vasopressin-sensitive adenylate cyclase activity (27, 33), whereas others were un- able to demonstrate this requirement (34, 35). Our data are compatible with the (lat- ter) view, that there was no minimal re- quirement for calcium since: (i) 1 mM EGTA enhanced both basal .and hormone- stimulated enzyme activity (Table I and Figs. 1 and 3); (ii) increasing calcium con- centration from 0.01 to 1 mM in the pres- ence of 1 InM EGTA (unpublished data) did not increase hormone sensitivity. However, a possible effect of EGTA by it- self, unrelated to its calcium chelating ability, cannot be ruled out (36). When a high calcium concentration (3 X 10e4 M or greater) was used, it inhibited renal med- ullary adenylate cyclase significantly. The inhibitory effect of calcium at high con- centrations has been reported in renal medullary preparations from hamster (5) and pig (27) as well as those derived from nonrenal tissues (37). It is difficult to de- rive a general principle from these varied effects of EGTA and calcium ions on renal adenylate cyclase obtained from different

FIG. 4. Restoration of bovine parathyroid hormone (bPTH)-1-34 sensitivity to particulate material (Ps) of dog renal middle medulla by GTP in the absence or presence of 1 rnM EGTA. Data represent means f SEM for five determinants in a single experiment. The concentration of bPTH-(1-34) employed in this experiment was 0.4 PM.

666 CHEN AND PUSCHETT

species. The variation in the effects of EGTA on the sensitivity of the renal en- zyme to vasopressin may well point to a complex enzyme system in the kidney.

On the basis of clearance studies per- formed in the dog, Martinez-Maldinado et al. have suggested that vasopressin may decrease the proximal reabsorption of so- dium and phosphate (38). The ADH effect on proximal phosphate transport was later verified in a micropuncture study by Wen (39) who demonstrated a decrease in prox- imal phosphate reabsorption after vaso- pressin administration to TPTX dogs. Fur- thermore, data from our laboratory have indicated that PTH in a high dosage (3-5 IU/kg) has an “ADH-like” effect on renal tubular water transport (40). More inter- estingly, it has been demonstrated that ADH may substitute for PTH as a “per- missive factor” for the antiphosphaturic effect of the vitamin D derivatives 25-OH-D3 and 1,25-(OH)zD3 to become manifest (41).

In a system containing two or more dif- ferent hormone receptors coupled to ade- nylate cyclase, simultaneous stimulation by hormones should induce an additive effect in enhancing CAMP formation. In the absence of exogenous calcium and the presence of 1 mM EGTA, an additive effect was observed in the cortex and middle medulla when AVP and PTH were added at their maximal doses. Such an additive effect has been reported by Nagata et aL (8) in renal papilla of rats stimulated by PTH and calcitonin. It is interesting to notice that their membrane fractions were obtained by homogenizing tissue in a buffer solution containing 1 mM EDTA. In the presence of endogenous calcium and the absence of EGTA, the PTH- and AVP- induced additive effect was completely eliminated (Tables II-IV). These data sug- gest that under this experimental circum- stance, both hormones were stimulating one single adenylate cyclase which was coupled to a common receptor for both hormones, or to two separate receptors, one for PTH and the other for AVP. Elim- ination of the EGTA-induced additive ef- fect by exogenous calcium suggests that the effect of EGTA is mediated, at least

in part, through its calcium chelating ability. Calcium ion concentration may therefore be critical in the modulation of the hormone-sensitive adenylate cyclase system.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of Dean Drosnes. Excellent secretarial assistance was provided by Candace Jones and Sharon Steliga. This work was supported (in part) by a grant from the National Science Foundation (PCM 77- 09655).

REFERENCES

1. ORLOFF, J., AND HANDLER, J. S. (1967) Amer. J. Med 42, 757-768.

2. CHASE, L. R., AND AURBACH, G. D. (1967) PTOC. Nat Acad Sci USA 58,518-525.

3. BROWN, E., CLARKE, D. L., Roux, V., AND SHER- MAN, G. H. (1962) J. Biol Chem 238, PC352- PC853.

4. CHASE, L. R., AND AURBACH, G. D. (1968) Scie7tce 159.545-547.

5. MARUMO, F., AND EDELMAN, I. S. (1971) J. Clin Invest. 50,1613-1620.

6. STREETO, J. (1969) MetuboZkm l&968-973. 7. DOUSA, T., HECHTER, O., SCHWARTZ, I. L., AND

WALTER, R. (1971) Proc Nat. Acad Sci. USA 68,1693-169’7.

8. NAGATA, N., ARAKI-SHIMADA, N., ONO, Y., AND KIMURA, N. (1978) Ada Endocrid 89, 404- 416.

9. CHABARDES, D., IYBERT, M., CLIQUE, A., MON- TEGUT, M., AND MOREL, F. (1975) PfEugers Arch 354,229-239.

10. IMBERT, M., CHABARDES, D., MONTEGUT, M., CLI- QUE, A., AND MOREL, F. (1975) P#ugers Arch 357,173-186.

11. CHABARDES, D., GAGNAN-BRUNETTE, M., IMBERT- TEBOIJL, M., GONTCHARVESKAI, O., MONTEGIJT, M., CLIQUE, A., AND MOREL, F. (1980) J. C&z. Invest. 65,439-448.

12. HEATH, D., AND AURBACH, G. D. (1974) in Cal- cium Regulating Hormones, Proceedings of the 5th Parathyroid Conference, pp. 159-162, Oxford.

13. NISSENSON, R., AND ARNAUD, C. D. (1979) J. Bid Chem 254,1469-1475.

14. SEGRE, G., ROSENBLA~, M., REINER, B., MAHAF- FEY, J. E., AND Porrs, J. T. JR. (1979) J. BzbL Chem 254,6980-6986.

15. BOCKAERT, J., ROY, C., RAJERISON, R., AND JARD, S. (1973) J. BioL Ch 248.5922-5931.

16. SCHRAMM, M., ORLY, J., EIMERL, J., AND KORNER, M. (1977) Nature (Zm&m) 268,310-313.

EFFECT OF Ca and GTP ON CANINE RENAL ADENYLATE CYCLASE 667

17. RODBELL, M., LIN, M. C., SOLOMON, Y., LONDOS, C., HARWOOD, J. P., MARTIN, B. R., RENDELL, M., AND BERMAN, M. (1975) in Advances in Cyclic Nucleotide Research, Vol. 5, pp. 3-29, Raven Press, New York.

18. FARFEL, Z., BRICKMAN, A. S., KASLOW, H. R., BROTHERS, V. M., AND BOURNE, H. R. (1980) N. EngL J. Med 303.237-242.

19. BRINBAUMER, L., AND RODBELL, M. (1969) J. Biol Chem. 244,3477-3482.

20. BAR, H. P., AND HECHTER, 0. (1969) Biochem Biophys. Res. Commun 35,681~686.

21. LEFKOWITZ, R. J., ROTH, J., AND PASTAN, I. (1970) Nature (London) 228, 864-866.

22. SUTCLIFFE, H. S., MARTIN, C. J., EISMAN, J. A., AND PILCZYK, R. (1973) Biochem. J. 134, 913- 921.

23. TRUMP, B. F., AND BULGER, R. E. (1968) in Struc- tural Basis of Renal Disease (Becker, E. L., ed.), pp. l-93, Harper & Row, New York.

24. CHEN, T. C., ROSENBLA?T, M., AND PUSCHE~, J. B. (1980) Biochem Biophys. Res. Commun 94. 122’7-1232.

25. BROWN, B. L., EKINS, R. P., AND ALBANO, J. D. M. (1972) in Advances in Cyclic Nucleo- tide Research, Vol. 2, pp. 25-40, Raven Press, New York.

26. LOWRY, 0. H., ROSEBROUGH, N. G., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Che.m 193, 265-275.

27. CAMPBELL, B., WOODWARD, G., AND BORBERG, V. (1972) J. BioL Chem 247, 6167-6175.

28. NEER, E. J. (1973) J. Bid Chem 248, 4775-4781.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

CHEN, T. C., COTE, T. E., AND KEBABIAN, J. W. (1980) Brain Res. 181,139-149.

COTE, T. E., CHEN, T. C., AND KEBABIAN, J. W. (1980) Brain Res. 181, 127-138.

CLARK, R. B. (1978) J. Cyclic Nuckotide Res. 4, 71-85.

HERMAN, C. A., ZENSER, T. V., AND DAVIS, B. B. (1978) Metabolism 27, 721-730.

BOCKAERT, J., ROY, C., AND JARD, S. (1972) J BioL Chem 247,7073-7081.

BAR, H. P., HECHTER, O., SCHWARTZ, I. L., AND WALTER, R. (1970) Proc. Nat. Acod. Sci. USA 67, 7-12.

DOUSA, T. P. (1972) Amer. J. PhysioL 222, 657- 662.

KEBABIAN, J. W. (1977) in Advances in Cyclic Nucleotide Research (Greengard, P., and Ro- bison, G. A., eds.), Vol. 8, pp. 421-508, Raven Press, New York.

PERKINS, J. P. (1973) in Advances in Cyclic Nu- cleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 3, pp. l-64, Raven Press, New York.

MARTINEZ-MALDONADO, M., EKNOYAN, G., AND SUKI, W. M. (1971) Amer. J. PhysioL 220,2013- 2020.

WEN, S. F. (1974) J. Clin. Invest. 53, 660-664.

FRAGOLA, J., WINAVER, J., ROBERTSON, G., CHEN, T. C., AND PUSCHETP, J. B. (1980) Clin Res. 28, 445A.

PUSCHETT, J. B., AND KUHRMAN, M. S. (1978) J. Lab. Clin Med. 92, 895-903.