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Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels
Life Sciences, Vol. 32, pp. 1009-1014 P~inted in the U.S.A.
Pergamon Pres~
PARATHYROID HORMONE STIMULATES ADENYLATE CYCLASE IN RAT CEREBRAL MICROVESSELS Minta Huang, D.A. Hanley and O.P. Rorstad Department of Medicine University of Calgary 3330 Hospital Drive N.W. Calgary, Alberta, Canada T2N 4NI (Received in final form November 16, 1982) Summary We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(I-84), the synthetic amino-terminal fragment bPTH-(I-34) and the synthetic analog [NIeS,NIelS,Tyr34]-bPTH- (i-34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent EDs0 values of 16 nM, 6.3 oM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle ~,NleI 8,Tyr34] -bPTH-(3-34) amide inhibited the action of bPTH-(I-84) and bPTH-(1-34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex. In addition to its principal action as a regulator of calcium metabolism, parathyroid hormone (PTH) shows biological effects on the heart (i) and blood vessels (2,3). Pang and colleagues produced hypotension in rats and dogs by infusion of bovine (b) PTH or bPTH-(1-34), a synthetic fragment consisting of the N-terminal 34 residues of bPTH (2). Studies of individual vascular beds have demonstrated vasodilation by PTH in the heart, kidney, liver, pancreas and stomach (3). The responsiveness of the cerebral vasculature to PTH has not been studied as yet. Cerebral cortical microvessels isolated from the rat and guinea pig possess adenylate cyclase activity which can be stimulated by a number of vasodilatory agents such as beta adrenergic agonists, prostaglandins, adenosine and vasoactive intestinal polypeptide (4,5). Because PTH dilates several vascular beds and PTH action on calcium homeostasis is mediated via activation of adenylate cyclase, the present study was undertaken to examine whether PTH would affect adenylate cyclase in rat cerebral microvessels. Materials and Methods [2,83H]cyclic AMP (36 Ci/mmol) was purchased from New England Nuclear (Boston, MA) and [=~32p] ATP (30-50 Ci/mmol) was from Amersham (Arlington Heights, IL). Native bPTH-(I-84) was obtained from Inolex Pharmaceuticals (Park Forest South, IL, Lot GO09, "highly purified", 1227 units/mg). Synthetic fragments of PTH were purchased from Beckman Instruments (Palo Alto, CA): bPTH-(I-34) (lots E1220, 6000 I.U./mg and BI0845, 6800 I.U./mg) and from Peninsula Laboratories (San Carlos, CA) : [Nle 8,Nle I 8 ,TyrS~] -bPTH(i-34) amide (lot 002746), [NleS,Nle 18,Tyr3~]-bpTH-(3-34) amide (lot 002410) and human (h) PTH-(13-34) (lot 002872). Phentolamine HCI was a gift from Dr. E. Vos at CIBA-Geigy Canada Ltd. (Mississauga, Ontario). Other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). 0024-3205 / 83/091009-06 $03. O0/0 Copyright (c) 1983 Pergamon Press Ltd.
i010
PTH: Microvessel Adenylate
Cyclase
Vol. 32, No. 9, 1983
Assay of a d e n x l a t e cyclase in cerebral microvessels. Cerebral microvessels were isolated from male Sprague-Dawley rats (275-350 g) according to the procedure of Huang and Drummond (5). Isolated microvessels were suspended in 20 mM Tris-HCl buffer (pH 7.5), 0.25 M sucrose and 2 mM dithiothreitol to give a protein concentration of 0.5-0.7 mg/ml and were stored at -70°C. The adenylate cyclase assay contained, in a final volume of 150 DI, 40 mM Tris-HCl buffer (pH 7.5), 1 mM 3-isobutyl-l-methylxanthine, i0 m~ MgSO4, 5.5 mM KCI, 10 mM phosphocreatine, 60 ~g creatine phosphokinase, 0.6 U adenosine deaminase, 0.1% bovine serum albumin (essentially fatty acid free), 0.4 mM dithiothreitol, 50 mM sucrose, 15-25 ~ g microvessel protein, 0.5 mM [~-32p]ATP (40-50 cpm/pmol) and test substance(s). Unless otherwise stated, guanosine triphosphate (GTP) was present in the assay at i0 UM. The assay components, except for [~_32p]ATP, were equilibrated at 30 °C for 1 min after which the substrate was added. The reaction was allowed to proceed at 30°C for i0 min and terminated b Y 2 h e a t inactivation of the enzyme at 80 ° C for 5 min. Quantitation of [3 P]cyclic AMP was by the method of Salomon et al (6). All assays were performed in duplicate and adenylate cyclase activity was expressed as pmol of cyclic AMP formed per mg microvessel protein per min. All duplicate determinations within an experiment differed by less than 10%. The results of each replicate experiment differed by less than 10%. Protein contents of microvessel preparations were determined by the method of Lowry e t al (7). Results Effects of PTH and its congeners. The basal adenylate cyclase activity of rat cerebral microvessel preparations was 37.9 ± 1.8 pmol cyclic AMP/mg protein/min (mean ± S ~ , n=12). Native bPTH-(I-84) caused a dose-related activation with an apparent ED50 of 16 nM (Fig. i). Synthetic bPTH-(I-34) was more potent than the native hormone. A small but statistically significant activation was seen with I nM of bPTH-(I-34) (Student's t-test, p< 0.001) and about 8-fold stimulation was achieved with i ~ M of this hormone fragment (ED50 = 6.3 nM). Two lots of bPTH-(I-34) demonstrated similar activity. The time course of activation by bPTH-(I-34) is shown in Fig. 2. The synthetic fragment alone, without exogenously added GTP in the assay, elicited a slight activation although addition of i0 ~M GTP greatly enhanced its action (Fig.
2). Analogs with amino acid deletions at the N-terminus of PTH, such as [NIe8,NIelB,Tyr34]-bPTH-(3-34) amide and hPTH-(13-34), were completely devoid of agonist activity even at 2 ~M (Fi$. I)~ The substituted hormone fragment with an intact PTH N-terminus, [NIe°,NIe~8,Tyr34]-bPTH-(I-34) amide, showed similar activity to bPTH-(1-34) with an apparent ED50 of 15 nM (Fig. 1). Inhibition of PTH action. The PTH inhibitor, [NleS,Nle I 8,Tyr34 ]_bPTH_ (3-34) amide, reduced the action of bPTH-(I-84) by 36% when both were present at equal molar concentration. However, this 3-34 substituted fragment inhibited the action of bPTH-(I-34) by 40% only when the former peptide was present at I0 times higher concentration (Table I). hPTH-(13-34) tested at molar ratios of 20:1 against hPTH-(I-34) or 2:1 against bPTH-(I-84) did not inhibit the stimulation caused by either bPTH-(I-34) or bPTH-(I-84).
Vol. 32, No. 9, 1983
PTH: Microvessel Adenylate Cyclase
1011
FIG. I. Stimulation of mlcrovessel adenylate cyclase by native bPTH and congeners of PTH.
(9)
3 o x
Adeny]ate cyclase activity was measured in the basal state (clear bar), or in the presence of varying concentrations of bPTH-(I-84) ([~), bPTH-(I-34) ( O ) , [Nle 8,Nle I 8, Tyr 34] -bPTH-(I-34) amide ( A ) , [ Nle 8 ,Nle I 8, Tyr3~ ] -bPTH-(3-34) amide (~) or hPTH-(13-34) (O). Each data point is the mean of two to four experiments except where otherwise indicated by the number in brackets. The vertical lides indicate the SEM.
E
2 uJ o3
_J >uJ F< I J >z w a
0
~
~
i
I
i
J
i
10
9
8
7
6
- log
[M]
FIG. 2.
/9
Basal and PTH-stimulated activities were measured in the presence ([~, O ) or absence (• ,A) of GTP (I0 pM). Each data point is the mean of two or three experiments. The vertical lines indicate the SEM.
.E E -.
Time course of activation of microvessel adenylate cyclase.
3
I
o
E
~2
>o I
/
A~[~]//-'/--BASAL(n°GTP) 5 TIME(mIn)
10
15
1012
PTH: Microvessel Adenvlate Cyc]ase
Vol. 32, No. 9, 1983
TABLE I
Effect of a PTH Inhibitor on the Action of Native bPTH-(I-34) on Microvessel Adenylate Cyclase. [Nle ~,Nle I ~ ,Tyr-{~+]beTH- (3-34) amide M 10-9 10-8 10-7 10-6
bPTH-(I-84)
and
% inh~ibition bPTH-_(I-84) bPTH-(I-34) i0 ~M 10-TM 0 11.9 24.5 36.4
0 0 11.6 40.0
Adenylate cyclase activity was measured with i0 -~ M native bPTH-(I-84) or 10-TM b-PTH-(I-34) plus increasing concentrations of ~leS,Nle 18, Tyr 34]-bPT~I-(3-34) amide. Values are the means from two separate experiments.
Effect of Adrenergic Adenylate Cyclase.
TABLE II Agents on the
of
bPTH-(I-34)
on
Adenylate cyclase (pmol cAMP/_m$/min) Control bPTH-(I-34) 0.i ~ M
Agents (~M)
None Phentolamine Propranolol Isoproterenol
Action
-I0 5 i0
32.4 34.7 34.0 87.3
270.5 266.7 278.9 346.3
Results are the averages from two experiments. Isoproterenol i0 ~ M was previously shown to elicit the maximal stimulation of adenylate cyclase capable by this agent (ref. 4). Propranolol ( 5 ~M) completely inhibited adenylate cyclase stimulation by i0 ~M isoproterenol. Interaction between bPTH-(I-34~ and adrener$ic agents. Neither phentolamine nor propranolol at much higher molar concentrations than bPTH-(I-34) affected its stimulatory action. The action of maximal stimulatory dose of isoproterenol (I0 DM, Ref. 4) and bPTH-(I-34) tested together approximated the sum of the stimulations elicited by the individual agonists (Table 2). Discussion The present study demonstrates that adenylate cyclase in microvessels isolated from rat cerebral cortex is responsive to stimulation by native bPTH-(I-84) and by the synthetic fragment bPTH-(I-34) which contains the amino acid sequence required for the known biological activity of the hormone (8). The apparent ED50 values for these two peptides are comparable to those
Vol. 32, No. 9, 1983
PTH: Microvessel Adenylate Cyclase
1013
observed in studies of canine renal cortical membranes (9), cultured bone tumour cells, and cultured skin fibroblasts (i0) (IG nM range). However, [Nle8,Nle18,Ty~]-bPTH-(l-34) amide, which is more potent than bPTH-(I-34) in stimulating adenylate cyclase in canine renal cortical membranes (11) was slightly less potent than bPTH-(I-34) in cerebral microvessels. Synthetic fragments of PTH lacking the N-terminal amino acid residues showed no agonist activity, which is consistent with previous studies using canine renal cortical membranes (12). Other studies have shown that the 3-34 fragment of PTH inhibits PTHstimulated adenylate cyclase in canine renal cortical membraoes (12) and cultured bone tumour cells (10). We observed that this analog inhibited the action of bPTH-(I-84) slightly less effectively than it did in the former studies. This difference may reflect intrinsic properties of tissue receptors or may be due to methodological factors. Addition of GTP to the microvessel preparation greatly enhanced the stimulation of adenylate cyclase by bPTH-(I-34). A low level of PTHstimulated activity in the absence of added GTP probably was due to the presence of a trace amount of endogenous GTP in the microvessel preparation. The PTH-responsive microvessel adenylate cyclase bears resemblance to other well described adenylate cyclases such as the beta adrenergic system, in that a guanine nucleotide binding component is required for activation of the enzyme by agonists ( 1 3 ) . However, our data indicate that PTH action on microvessel adenylate cyclase is clearly not mediated via the alpha or beta adrenergic receptor, because neither phentolamine nor propranolol inhibited the stimulatory action of bPTH-(1-34). Furthermore, the combined effect of maximal stimulatory doses of isoproterenol (i0 ~ M) and bPTH-(I-34) approximated the sum of the two agents tested separately, suggesting that distinct receptors are present for the two ligands. The results of this study and the previous observations by other workers that PTH dilates several vascular beds (2,3) provoke the question whether PTH may play a physiologically significant role in the regulation of cerebral blood flow. The ED50for the most potent adenylate cyclase activator which we had previously studied using this system, vasoactive intestinal polypeptide, was I00 nM (4). PTH was clearly more potent with an ED50 in the range of 6.3 to 16 nM. The EDs0 for PTH activation of adenylate cyclase in renal membranes is also approximately i0 nM and this activation has been widely recognized as being a mechanism underlying the physiological action of PTH. In addition, the magnitude of stimulation of cerebral microvessel adenylate cyclase by PTH was far greater than that effected by any other vasoactive agent we have tested (c.f. Ref. 4). As yet there has been no report of PTH action on cerebral vascular tone or blood flow. In conclusion, the data presented herein indicate that PTH is a potent stimulator of adenylate cyclase in rat cerebral microvessels and thereby merits further investigation with respect to its potential for the regulation of cerebral blood flow. Acknowledgements This study was supported by the Alberta Heritage Foundation for Medical Research, the Medical Research Council of Canada and the Alberta Mental Health Advisory Council. The authors thank Wendy Hoopfer for secretarial assistance. References i.
F. LHOSTE, T. DRUEKE, S. LARNO and J.R. BOSSIER, Physiol. 7 119-127 (1980).
Clln.
Exp. Pharmacol.
1014
2. 3. 4. 5. 6. 7. 8.
9. i0. ii. 12. 13.
PTH: Microvessel Adenylate Cyclase
Vol.
32, No. 9, 1983
P.K.T. PANG, T.E. TENNER, JR., J.A. YEE, M. YANG and H.F. JANSSEN, Proc. Natl. Acad. Sci. U.S.A. 77 675-678 (1980). P.K.T. PANG, H.F. JANSSEN and J.A. YEE, Pharmacology 2 1 213-222 (1980). M. HUANG and O.P. RORSTAD, J. Neurochem. (in press). M. HUANG and G.I. DRU~IMOND, Mol. Pharmacol. 16 462-472 (1979). Y. SALOMON, C. LONDOS and M. RODBELL, Anal. Biochem. 58 541-548 (1974). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). J.T. POTTS, JR., G.W. T R E G E ~ , H.T. KEU~MANN, H.D. NIALL, R. SAUER, L.J. DEFTOS, B.F. DAWSON, M.L. HOGAN and G.D. AURBACH, Proc. Natl. Acad. Sci. U.S.A. 68 63-67 (1971). D. GOLTZMAN, E.N. CALLAHAN, G.W. TREGEAR and J.T. POTTS, JR. , Endocrinology 103 1352-1360 (1978). S.R. GOLDRING, J.E. MAHAFFEY, M. ROSENBLATT, J.M. DAYER, J.T. POTTS, JR. and S.M. KRANE, J. Clin. Endocrinol. Metab. 48 655-659 (1979). G.V. SEGRE, M. ROSENBLATT, B.L. REINER, J.E. MAHAFFEY and J.T. POTTS, JR., J. Biol. Chem 254 6980-6986 (1979). M. ROSENBLATT, E.N. CALLAHAN, J.E. MAHAFFEY, A. PONT and J.T. POTTS, JR., J. Biol. Chem. 252 5847-5851 (1977). E.M. ROSS and A.G. GILMAN, Annu. Rev. Biochem. 49 533-564 (1980).
Pergamon Pres~
PARATHYROID HORMONE STIMULATES ADENYLATE CYCLASE IN RAT CEREBRAL MICROVESSELS Minta Huang, D.A. Hanley and O.P. Rorstad Department of Medicine University of Calgary 3330 Hospital Drive N.W. Calgary, Alberta, Canada T2N 4NI (Received in final form November 16, 1982) Summary We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(I-84), the synthetic amino-terminal fragment bPTH-(I-34) and the synthetic analog [NIeS,NIelS,Tyr34]-bPTH- (i-34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent EDs0 values of 16 nM, 6.3 oM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle ~,NleI 8,Tyr34] -bPTH-(3-34) amide inhibited the action of bPTH-(I-84) and bPTH-(1-34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex. In addition to its principal action as a regulator of calcium metabolism, parathyroid hormone (PTH) shows biological effects on the heart (i) and blood vessels (2,3). Pang and colleagues produced hypotension in rats and dogs by infusion of bovine (b) PTH or bPTH-(1-34), a synthetic fragment consisting of the N-terminal 34 residues of bPTH (2). Studies of individual vascular beds have demonstrated vasodilation by PTH in the heart, kidney, liver, pancreas and stomach (3). The responsiveness of the cerebral vasculature to PTH has not been studied as yet. Cerebral cortical microvessels isolated from the rat and guinea pig possess adenylate cyclase activity which can be stimulated by a number of vasodilatory agents such as beta adrenergic agonists, prostaglandins, adenosine and vasoactive intestinal polypeptide (4,5). Because PTH dilates several vascular beds and PTH action on calcium homeostasis is mediated via activation of adenylate cyclase, the present study was undertaken to examine whether PTH would affect adenylate cyclase in rat cerebral microvessels. Materials and Methods [2,83H]cyclic AMP (36 Ci/mmol) was purchased from New England Nuclear (Boston, MA) and [=~32p] ATP (30-50 Ci/mmol) was from Amersham (Arlington Heights, IL). Native bPTH-(I-84) was obtained from Inolex Pharmaceuticals (Park Forest South, IL, Lot GO09, "highly purified", 1227 units/mg). Synthetic fragments of PTH were purchased from Beckman Instruments (Palo Alto, CA): bPTH-(I-34) (lots E1220, 6000 I.U./mg and BI0845, 6800 I.U./mg) and from Peninsula Laboratories (San Carlos, CA) : [Nle 8,Nle I 8 ,TyrS~] -bPTH(i-34) amide (lot 002746), [NleS,Nle 18,Tyr3~]-bpTH-(3-34) amide (lot 002410) and human (h) PTH-(13-34) (lot 002872). Phentolamine HCI was a gift from Dr. E. Vos at CIBA-Geigy Canada Ltd. (Mississauga, Ontario). Other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). 0024-3205 / 83/091009-06 $03. O0/0 Copyright (c) 1983 Pergamon Press Ltd.
i010
PTH: Microvessel Adenylate
Cyclase
Vol. 32, No. 9, 1983
Assay of a d e n x l a t e cyclase in cerebral microvessels. Cerebral microvessels were isolated from male Sprague-Dawley rats (275-350 g) according to the procedure of Huang and Drummond (5). Isolated microvessels were suspended in 20 mM Tris-HCl buffer (pH 7.5), 0.25 M sucrose and 2 mM dithiothreitol to give a protein concentration of 0.5-0.7 mg/ml and were stored at -70°C. The adenylate cyclase assay contained, in a final volume of 150 DI, 40 mM Tris-HCl buffer (pH 7.5), 1 mM 3-isobutyl-l-methylxanthine, i0 m~ MgSO4, 5.5 mM KCI, 10 mM phosphocreatine, 60 ~g creatine phosphokinase, 0.6 U adenosine deaminase, 0.1% bovine serum albumin (essentially fatty acid free), 0.4 mM dithiothreitol, 50 mM sucrose, 15-25 ~ g microvessel protein, 0.5 mM [~-32p]ATP (40-50 cpm/pmol) and test substance(s). Unless otherwise stated, guanosine triphosphate (GTP) was present in the assay at i0 UM. The assay components, except for [~_32p]ATP, were equilibrated at 30 °C for 1 min after which the substrate was added. The reaction was allowed to proceed at 30°C for i0 min and terminated b Y 2 h e a t inactivation of the enzyme at 80 ° C for 5 min. Quantitation of [3 P]cyclic AMP was by the method of Salomon et al (6). All assays were performed in duplicate and adenylate cyclase activity was expressed as pmol of cyclic AMP formed per mg microvessel protein per min. All duplicate determinations within an experiment differed by less than 10%. The results of each replicate experiment differed by less than 10%. Protein contents of microvessel preparations were determined by the method of Lowry e t al (7). Results Effects of PTH and its congeners. The basal adenylate cyclase activity of rat cerebral microvessel preparations was 37.9 ± 1.8 pmol cyclic AMP/mg protein/min (mean ± S ~ , n=12). Native bPTH-(I-84) caused a dose-related activation with an apparent ED50 of 16 nM (Fig. i). Synthetic bPTH-(I-34) was more potent than the native hormone. A small but statistically significant activation was seen with I nM of bPTH-(I-34) (Student's t-test, p< 0.001) and about 8-fold stimulation was achieved with i ~ M of this hormone fragment (ED50 = 6.3 nM). Two lots of bPTH-(I-34) demonstrated similar activity. The time course of activation by bPTH-(I-34) is shown in Fig. 2. The synthetic fragment alone, without exogenously added GTP in the assay, elicited a slight activation although addition of i0 ~M GTP greatly enhanced its action (Fig.
2). Analogs with amino acid deletions at the N-terminus of PTH, such as [NIe8,NIelB,Tyr34]-bPTH-(3-34) amide and hPTH-(13-34), were completely devoid of agonist activity even at 2 ~M (Fi$. I)~ The substituted hormone fragment with an intact PTH N-terminus, [NIe°,NIe~8,Tyr34]-bPTH-(I-34) amide, showed similar activity to bPTH-(1-34) with an apparent ED50 of 15 nM (Fig. 1). Inhibition of PTH action. The PTH inhibitor, [NleS,Nle I 8,Tyr34 ]_bPTH_ (3-34) amide, reduced the action of bPTH-(I-84) by 36% when both were present at equal molar concentration. However, this 3-34 substituted fragment inhibited the action of bPTH-(I-34) by 40% only when the former peptide was present at I0 times higher concentration (Table I). hPTH-(13-34) tested at molar ratios of 20:1 against hPTH-(I-34) or 2:1 against bPTH-(I-84) did not inhibit the stimulation caused by either bPTH-(I-34) or bPTH-(I-84).
Vol. 32, No. 9, 1983
PTH: Microvessel Adenylate Cyclase
1011
FIG. I. Stimulation of mlcrovessel adenylate cyclase by native bPTH and congeners of PTH.
(9)
3 o x
Adeny]ate cyclase activity was measured in the basal state (clear bar), or in the presence of varying concentrations of bPTH-(I-84) ([~), bPTH-(I-34) ( O ) , [Nle 8,Nle I 8, Tyr 34] -bPTH-(I-34) amide ( A ) , [ Nle 8 ,Nle I 8, Tyr3~ ] -bPTH-(3-34) amide (~) or hPTH-(13-34) (O). Each data point is the mean of two to four experiments except where otherwise indicated by the number in brackets. The vertical lides indicate the SEM.
E
2 uJ o3
_J >uJ F< I J >z w a
0
~
~
i
I
i
J
i
10
9
8
7
6
- log
[M]
FIG. 2.
/9
Basal and PTH-stimulated activities were measured in the presence ([~, O ) or absence (• ,A) of GTP (I0 pM). Each data point is the mean of two or three experiments. The vertical lines indicate the SEM.
.E E -.
Time course of activation of microvessel adenylate cyclase.
3
I
o
E
~2
>o I
/
A~[~]//-'/--BASAL(n°GTP) 5 TIME(mIn)
10
15
1012
PTH: Microvessel Adenvlate Cyc]ase
Vol. 32, No. 9, 1983
TABLE I
Effect of a PTH Inhibitor on the Action of Native bPTH-(I-34) on Microvessel Adenylate Cyclase. [Nle ~,Nle I ~ ,Tyr-{~+]beTH- (3-34) amide M 10-9 10-8 10-7 10-6
bPTH-(I-84)
and
% inh~ibition bPTH-_(I-84) bPTH-(I-34) i0 ~M 10-TM 0 11.9 24.5 36.4
0 0 11.6 40.0
Adenylate cyclase activity was measured with i0 -~ M native bPTH-(I-84) or 10-TM b-PTH-(I-34) plus increasing concentrations of ~leS,Nle 18, Tyr 34]-bPT~I-(3-34) amide. Values are the means from two separate experiments.
Effect of Adrenergic Adenylate Cyclase.
TABLE II Agents on the
of
bPTH-(I-34)
on
Adenylate cyclase (pmol cAMP/_m$/min) Control bPTH-(I-34) 0.i ~ M
Agents (~M)
None Phentolamine Propranolol Isoproterenol
Action
-I0 5 i0
32.4 34.7 34.0 87.3
270.5 266.7 278.9 346.3
Results are the averages from two experiments. Isoproterenol i0 ~ M was previously shown to elicit the maximal stimulation of adenylate cyclase capable by this agent (ref. 4). Propranolol ( 5 ~M) completely inhibited adenylate cyclase stimulation by i0 ~M isoproterenol. Interaction between bPTH-(I-34~ and adrener$ic agents. Neither phentolamine nor propranolol at much higher molar concentrations than bPTH-(I-34) affected its stimulatory action. The action of maximal stimulatory dose of isoproterenol (I0 DM, Ref. 4) and bPTH-(I-34) tested together approximated the sum of the stimulations elicited by the individual agonists (Table 2). Discussion The present study demonstrates that adenylate cyclase in microvessels isolated from rat cerebral cortex is responsive to stimulation by native bPTH-(I-84) and by the synthetic fragment bPTH-(I-34) which contains the amino acid sequence required for the known biological activity of the hormone (8). The apparent ED50 values for these two peptides are comparable to those
Vol. 32, No. 9, 1983
PTH: Microvessel Adenylate Cyclase
1013
observed in studies of canine renal cortical membranes (9), cultured bone tumour cells, and cultured skin fibroblasts (i0) (IG nM range). However, [Nle8,Nle18,Ty~]-bPTH-(l-34) amide, which is more potent than bPTH-(I-34) in stimulating adenylate cyclase in canine renal cortical membranes (11) was slightly less potent than bPTH-(I-34) in cerebral microvessels. Synthetic fragments of PTH lacking the N-terminal amino acid residues showed no agonist activity, which is consistent with previous studies using canine renal cortical membranes (12). Other studies have shown that the 3-34 fragment of PTH inhibits PTHstimulated adenylate cyclase in canine renal cortical membraoes (12) and cultured bone tumour cells (10). We observed that this analog inhibited the action of bPTH-(I-84) slightly less effectively than it did in the former studies. This difference may reflect intrinsic properties of tissue receptors or may be due to methodological factors. Addition of GTP to the microvessel preparation greatly enhanced the stimulation of adenylate cyclase by bPTH-(I-34). A low level of PTHstimulated activity in the absence of added GTP probably was due to the presence of a trace amount of endogenous GTP in the microvessel preparation. The PTH-responsive microvessel adenylate cyclase bears resemblance to other well described adenylate cyclases such as the beta adrenergic system, in that a guanine nucleotide binding component is required for activation of the enzyme by agonists ( 1 3 ) . However, our data indicate that PTH action on microvessel adenylate cyclase is clearly not mediated via the alpha or beta adrenergic receptor, because neither phentolamine nor propranolol inhibited the stimulatory action of bPTH-(1-34). Furthermore, the combined effect of maximal stimulatory doses of isoproterenol (i0 ~ M) and bPTH-(I-34) approximated the sum of the two agents tested separately, suggesting that distinct receptors are present for the two ligands. The results of this study and the previous observations by other workers that PTH dilates several vascular beds (2,3) provoke the question whether PTH may play a physiologically significant role in the regulation of cerebral blood flow. The ED50for the most potent adenylate cyclase activator which we had previously studied using this system, vasoactive intestinal polypeptide, was I00 nM (4). PTH was clearly more potent with an ED50 in the range of 6.3 to 16 nM. The EDs0 for PTH activation of adenylate cyclase in renal membranes is also approximately i0 nM and this activation has been widely recognized as being a mechanism underlying the physiological action of PTH. In addition, the magnitude of stimulation of cerebral microvessel adenylate cyclase by PTH was far greater than that effected by any other vasoactive agent we have tested (c.f. Ref. 4). As yet there has been no report of PTH action on cerebral vascular tone or blood flow. In conclusion, the data presented herein indicate that PTH is a potent stimulator of adenylate cyclase in rat cerebral microvessels and thereby merits further investigation with respect to its potential for the regulation of cerebral blood flow. Acknowledgements This study was supported by the Alberta Heritage Foundation for Medical Research, the Medical Research Council of Canada and the Alberta Mental Health Advisory Council. The authors thank Wendy Hoopfer for secretarial assistance. References i.
F. LHOSTE, T. DRUEKE, S. LARNO and J.R. BOSSIER, Physiol. 7 119-127 (1980).
Clln.
Exp. Pharmacol.
1014
2. 3. 4. 5. 6. 7. 8.
9. i0. ii. 12. 13.
PTH: Microvessel Adenylate Cyclase
Vol.
32, No. 9, 1983
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