- Email: [email protected]
Antilipolytic activity of viprostol, a transdermally active antihypertensive PGE2 analog
PROSTAGLANDINS
ANTILIPOLYTIC ACTIVITY OF VIPROSTOL, A TRANSDERMALLY ACTIVE ANTIHYPERTENSIVE PGE 2 ANALOG David L. Crandall, VanessaVu, Florence H. Lizzo, Barbara A. Davis and Peter Cervoni Dept. Cardiovascular Biological Research, American Cyanamid Co., Medical Research Div., Lederle Laboratories, Pearl River, NY 10965 ABSTRACT Viprostol, a novel prostaglandin E_ congener, was assessed for in vitro antilipolytic activity in the spontaneously obese rat. In--iso~ed epididymal adipocytes, viprostol exhibited a dose-dependent inhibition of catecholamine-stimulated lipolysis at concentrations ranging from i0 ~M to 1 mM, but was ineffective at lower concentrations. Additionally, viprostol exhibited approximately 50% of the antilipolytic activity of naturally-occurring PGE I and PGE_ at similar concentrations, but was as potent as PGF.. At i0 ~ , viprostol inhibited maximum catecholamlne-stlmula~ed lipolysis by approximately 35% of the total, hormone-stimulated glycerol release. The results of these experiments indicate that viprostol exhibits antilipolytic activity in vitro, but is less potent than the naturallyoccurring PGE's to which it is most closely related structurally. INTRODUCTION Prostaglandins (PG) have been known to influence certain aspects of lipid metabolism since reports of the antilipolytic activity of PGE described over two decades ago (i). More recently, PGE binding sites have been well characterized on adipocyte membranes (2), ultimately providing additional support to the hypothesis that PGE's are true endogenous modulators of lipolysis. While control of lipolysis through external administration of PG could be beneficial for certain clinical disorders (3), this realization has been hindered in part by the short biological half-lives of these hormones. Many investigators have, therefore, searched for synthetic alterations in the structures of naturally-occurring PG that would result in analogues with biological activities similar to the natural hormones, but which are not rapidly catabolized. Interestingly, our laboratory has recently reported the discovery of a synthetic PGE 2 analog, viprostol, which exhibits a profile of long biological activity in animal models of cardiovascular disease (4). Specifically, this compound exhibits antihypertensive activity following a single administration by either the oral, intravenous, or transdermal route, and the hypotensive response is of a relatively long duration (>24 hr). At present, however, no reports exist concerning the anti-
MAY 1987 VOL. 33 NO. 5
767
PROSTAGLANDINS
lipolytic activity of this compound. The present study has therefore investigated the antilipolytic activity of viprostol in an attempt to determine whether this compound affects lipid metabolism in a manner similar to the naturally-occurring PGE's. METHODS
Spontaneously obese male Sprague-Dawley rats were killed in the fed state by cervical dislocation on the morning of the experiment, and isolated adipocytes were prepared according to the method of Rodbell (5), using modifications described previously (6). Washed fat cells were then incubated (0.5xl06/flask) in a final volume of 2 ml Kreb's Ringer bicarbonate (KRB) buffer containing 6 mM glucose and maximally stimulating concentrations of 1-epinephrine bitartrate (10-5M). The incubations were initiated by the addition of fat cells, followed by gassing (95% 02-5% CO.) and capping of the vials. All vials were incubated in triplica£e for 60 min at 37°C in a metabolic shaker set at 60 strokes/min. The incubations were subsequently terminated and the medium was recovered (6). Preliminary experiments confirmed that lipolysis was linear during the incubation period (n=3). Viprostol (CL 115,347; dl-15-deoxy-16-hydroxy-16(~/6)vinyl PGE2) was synthesized and formulated as described previously (~,7). To determine the relative inhibition of catecholamine-stimulated lipolysis, 20 ~i of viprostol in ethanol was added to the incubations in triplicate to achieve a final concentration of 10-3 to 10-7M, and in the presence of epinephrine. For the determination of the comparative antilipolytic activity, either viprostol or a naturally-occurring prostaglandin (PGEI, PGE. or PGF. ; Ono Pharmaceutical Co., Osaka, Japan) was added £o the a~pocyte incubation in the presence of epinephrine. Each was dissolved in absolute ethanol, and added in a volume of 20 ~i at a final concentration of 10 ~M. Preliminary experiments indicated that the ethanol vehicle alone did not affect the results. Following the assessment of total medium glycerol (8), fat cell size and number were calculated (9), ultimately allowing the expression of lipolysis per adipocyte. The relative capability of each PG to inhibit catecholamine-stimulated lipolysis was calculated by comparing the absolute glycerol in PG-containing vials to those containing only the catecholamine (100% lipolysis). Values from each group were expressed as the mean ± the standard error of the mean (SEM), and were considered significantly different at p<.05. Linear regression analysis was used to determine the effective concentration at which 50% of the hormone-stimulated lipolysis wouldbe inhibited by viprostol (10). Mean values were compared using the t-test.
768
MAY 1987 VOL. 33 NO. 5
PROSTAGLANDINS
RESULTS Viprostol exhibited a dose-dependent inhibition of catecholamine-stimulated lipolysis (Table i). In isolated adipocytes, maximally stimulating concentrations of epinephrine (i0 M) alone resulted in over a 30x increase above basal lipolysis, viprostol was ineffective in the inhibition of hormone-stimulated lipolysis ~t 10-6 and 10-TM. A reduction in lipolysis was observed at i0- M, but significant inhibition in glycerol release was observed only at 10-3 and 10-4M. The inhibition of 50% of the observed catecholamine-stimulated lipolysis was estimated as 3.45x10-4M. -5
,
Table i. Concentration of Viprostol
(M) 10-3
.
Effect of Viprostol on CatecholamineStimulated Lipolysis Glycerol Release (~mol/106fc/hr) .354±.04**
i0 -4
1.16 ±.21"
10-5
2.07 ±.39
10-6
2.55 ±.35
10-7
2.59 ±.39
Basal Epinephrinestimulated
Adipocyte Characteristics Diameter: 75.4±2.6 microns Volume: 260±26 picoliters Number per flask: .436±.051 x 106
Rat body weight: 504±27 g
.071±.018 2.45 ±.36
Values are mean±SEM of 7 separate experiments with triplicate incubations for each concentration. * Significantly different from epinephrine-stimulated value at p<.05 and **p<.001. In a separate set of experiments, the comparative antilipolytic activity of viprostol and naturally-occurring PG's was assessed. As seen in Figure i, at equimolar concentrations (i0 ~M), PGE I and PGE~ inhibited lipolysis by approximately 65%, where catecholamine-s%imulated glycerol release was considered as 100% lipolysis. PGF2~ was the least potent of the naturally-
MAY 1987 VOL. 33 NO. 5
769
PROSTAGLANDINS
occurring PG's, while viprostol inhibited maximally induced lipolysis by approximately 37%. When tested individually, either viprostol or PGF2. was capable of significantly (p<.05) reducing epinephrine-stimulated glycerol release. However, the antilipolytic potency of PGEI or PGE2 was significantly greater than that of either viprostol or PGF2~, based upon a significant reduction (p<.025) of released ~lycerol in the presence of equimolar concentrations of either PGEI or PGE 2 compared directly to PGF. or viprostol. From these experiments, it is estimated that vlprostol exhibited approximately 50% of the activity of PGE in vitro. Figure I:
Comparative Effect of Viprostol (CL 115,347) in the Inhibition of CatecholamineStimulated Lipolysis
70
q_
60
50 r--1 40
PGE]
HIll]I]]] PGE 2
Relative Inhibition of L i p o l y s i s (%)
PGF2~
30
m
ll-
r~.-..-..-..'~ CL 115, 347
M_n..:'
20
10
0
Because of the maximally-stimulating concentrations of catecholamine used in these experiments, incubations including a methyl xanthine provided insight toward the integrity of the ~-adrenceptor. As shown in Table 2, IBMX significantly stimulated lipolysis, which was dramatically and significantly inhibited by 10 ~M viprostol. Since lipolysis due to IBMX remained intact, these data imply that viprostol acts distally to the ~-adrenoceptor.
770
MAY 1987 VOL. 33 NO. 5
PROSTAGLANDINS
Table 2.
Effect of Isobutyl-methyl-xanthine (IBMX) on Viprostol's Antilipolytic Activity
Incubation Condition
Glycerol Release (pmol/106 fc/hr)
Adipocyte Characteristics
Basal
1.20±.18
Diameter:
Basal + Viprostol
1.90±.23
108.4±3.4 microns
(i00 ~M) Epinephrine
Volume: 4.62±.66a
779±71 picoliters
Epinephrine + Viprostol
1.66±.08
Number per flask:
IBMX
8.35±1.55"
(i0 ~M)
.259±.034 x 106
(1 mM) IBMX + Viprostol
Rat Body Weight: 1.79±.25
722±46 g
Values are mean±SEM of 2-5 separate experiments, with each incubation condition in triplicate. No significant differences were apparent between Basal and Basal + Viprostol. a Significantly different from Basal-Viprostol value at p<.025. DISCUSSION These studies indicate that viprostol exhibits antilipolytic activity in vitro in a dose-dependent manner, but that the inhibition of lipolys-is was evident only at relatively high concentrations of the compound. In fact, other investigators have estimated the fiftypercent inhibition of catecholamine stimulated lipolysis for PGE 2 in the micromolar range, indicating an approximate i00 times greater potency than viprostol (3). We have usedmaximally stimulating concentrations of catecholamine, however, and the aforementioned report of relative potency of PG used a submaximal concentration of catecholamine. Importantly, the dose-response for isolated adipocyte release of glycerol following catecholamine stimulation is quite variable at sub-maximal concentrations (ii), and maximal stimulation has often been considered necessary when assessing lipolysis in isolated fat cells (6,11). The activity of viprostol in terms of the relative inhibition of lipolysis should therefore also be analyzed in comparison to the antilipolytic activity of naturally-occurring PG. The relative inhibition that we observed agrees well with published data using similar incubation conditions (i). From these data, it
MAY 1987 VOL. 33 NO. 5
771
PROSTAGLANDINS
appears that viprostol is approximately 50% as potent as PGE, and equally potent to PGF 2 . The antilipolytic potency of viprostol may also have been underestimated because, as indicated by the structural formula, viprostol is a mixture of multiple molecules. Preliminary observations in our laboratory indicate that the separated 1-isomer is approximately 4x more potent that viprostol with respect to antihypertensive activity. In summary, viprostol is a synthetic PGE_ analog in which structural alteratlons have resulted in a reductlon in antilipolytic activity when compared directly to the naturally-occurring hormone. While viprostol exhibits antilipolytic activity, the concentrations required to inhibit maximally-induced lipolysis in vitro are indicative of a diminution in antilipolytic activ-lty--~lowing movement of the hydroxyl group to the 16-carbon position. Interestingly, this compound is documented to produce other biological effects associated with PGE, and after a single oral or transdermal administration (4,12). In a similar manner, future experiments examining antilipolytic activity in vivo may therefore be used to better assess the effects of vipro-sto~n the regulation of lipid metabolism. REFERENCES i.
Steinberg, D., M. Vaughan, P. Nestel, O. Strand, and S. Bergstrom. Effects of the prostaglandins on hormoneinduced mobilization of free fatty acids. J. Clin. Invest. 43: 1533, 1964.
2.
Robertson, R.P., and S. Little. Down-regulation of prostaglandin E receptors and homologous desensitization in isolated adipocytes. Endocrinology 113: 1732, 1983.
3.
Axelrod, L., Trzepacz, P., Zusman, R., and Martin, D. Antilipolytic effect of prostaglandin E 2 analogues: therapeutic implications. Life Sci. 18: 627, 1976.
4.
Chan, P.S., P. Cervoni, M. Ronsberg, R. Accomando, G. Quirk, P. Scully, and L. Lipchuck. Antihypertensive activity of d,l-15-deoxy-16-hydroxy-16(~/8)-vinyl prostaglandin E 2 methyl ester (CL 115,347), a new orally and transdermally longacting antihypertensive agent. J. Pharmacol. Exp. Ther. 226: 726, 1983.
5.
Rodbell, M. Metabolism of isolated fat cells. Chem. 239: 375, 1964.
6.
Crandall, D.L., S. Fried, A. Francendese, M. Nickel, and M. DiGirolamo. Lactate release from isolated adipocytes: influence of cell size, glucose concentration, insulin and epinephrine. Horm. Metab. Res. 15: 326, 1983.
772
J. Biol.
MAY 1987 VOL. 33 NO. 5
PROSTAGLANDINS
7.
Birnbaum, J.E., P. Cervoni, P.S. Chan, S. Chen, M. Floyd, C. Grudzinskas, M. Weiss, and F. Dessy. Prostaglandins and congeners 29. (16RS)-±-15-deoxy-16-hydroxy-16-vinylprostaglandin E 2 , an orally and transdermally active hypotensive agent of prolonged duration. J. Med. Chem. 25: 492-4, 1982.
8.
Pinter, J.D., J. Hayashi, and J.A. Watson. Enzymatic assay of glycerol, dihydroxyacetone and glyceraldehyde. Arch. Biochem. Biophys. 121: 404, 1967.
9.
DiGirolamo, M., S. Mendlinger, and J. Fertig. A simple method to determine fat cell size and number in four ma~nalia species. Am. J. Physiol. 221: 850, 1971.
i0. Snedecor, G., and W. Cochran. Statistical Methods, 6th Ed. Iowa State Univ. Press, Ames, 1967. p. 549. ii. Zinder, O., and B. Shapiro. Effect of cell size on epinephrine- and ACTH-induced fatty acid release from isolated fat cells. J. Lipid Res. 12: 91-95, 1971. 12. Crandall, D.L., B.M. Goldstein, R. Gabel, F.H. Lizzo, and P. Cervoni. Effects of the antihypertensive prostaglandin analog CL 115,347 on cardiac output distribution in the spontaneously hypertensive rat. J. Cardiovasc. Pharmacol. 7: 996, 1985.
Editor: R.P. Robertson
MAY 1987 VOL. 33 NO. 5
Received:
9-17-86
Accepted:
3-30-87
773
ANTILIPOLYTIC ACTIVITY OF VIPROSTOL, A TRANSDERMALLY ACTIVE ANTIHYPERTENSIVE PGE 2 ANALOG David L. Crandall, VanessaVu, Florence H. Lizzo, Barbara A. Davis and Peter Cervoni Dept. Cardiovascular Biological Research, American Cyanamid Co., Medical Research Div., Lederle Laboratories, Pearl River, NY 10965 ABSTRACT Viprostol, a novel prostaglandin E_ congener, was assessed for in vitro antilipolytic activity in the spontaneously obese rat. In--iso~ed epididymal adipocytes, viprostol exhibited a dose-dependent inhibition of catecholamine-stimulated lipolysis at concentrations ranging from i0 ~M to 1 mM, but was ineffective at lower concentrations. Additionally, viprostol exhibited approximately 50% of the antilipolytic activity of naturally-occurring PGE I and PGE_ at similar concentrations, but was as potent as PGF.. At i0 ~ , viprostol inhibited maximum catecholamlne-stlmula~ed lipolysis by approximately 35% of the total, hormone-stimulated glycerol release. The results of these experiments indicate that viprostol exhibits antilipolytic activity in vitro, but is less potent than the naturallyoccurring PGE's to which it is most closely related structurally. INTRODUCTION Prostaglandins (PG) have been known to influence certain aspects of lipid metabolism since reports of the antilipolytic activity of PGE described over two decades ago (i). More recently, PGE binding sites have been well characterized on adipocyte membranes (2), ultimately providing additional support to the hypothesis that PGE's are true endogenous modulators of lipolysis. While control of lipolysis through external administration of PG could be beneficial for certain clinical disorders (3), this realization has been hindered in part by the short biological half-lives of these hormones. Many investigators have, therefore, searched for synthetic alterations in the structures of naturally-occurring PG that would result in analogues with biological activities similar to the natural hormones, but which are not rapidly catabolized. Interestingly, our laboratory has recently reported the discovery of a synthetic PGE 2 analog, viprostol, which exhibits a profile of long biological activity in animal models of cardiovascular disease (4). Specifically, this compound exhibits antihypertensive activity following a single administration by either the oral, intravenous, or transdermal route, and the hypotensive response is of a relatively long duration (>24 hr). At present, however, no reports exist concerning the anti-
MAY 1987 VOL. 33 NO. 5
767
PROSTAGLANDINS
lipolytic activity of this compound. The present study has therefore investigated the antilipolytic activity of viprostol in an attempt to determine whether this compound affects lipid metabolism in a manner similar to the naturally-occurring PGE's. METHODS
Spontaneously obese male Sprague-Dawley rats were killed in the fed state by cervical dislocation on the morning of the experiment, and isolated adipocytes were prepared according to the method of Rodbell (5), using modifications described previously (6). Washed fat cells were then incubated (0.5xl06/flask) in a final volume of 2 ml Kreb's Ringer bicarbonate (KRB) buffer containing 6 mM glucose and maximally stimulating concentrations of 1-epinephrine bitartrate (10-5M). The incubations were initiated by the addition of fat cells, followed by gassing (95% 02-5% CO.) and capping of the vials. All vials were incubated in triplica£e for 60 min at 37°C in a metabolic shaker set at 60 strokes/min. The incubations were subsequently terminated and the medium was recovered (6). Preliminary experiments confirmed that lipolysis was linear during the incubation period (n=3). Viprostol (CL 115,347; dl-15-deoxy-16-hydroxy-16(~/6)vinyl PGE2) was synthesized and formulated as described previously (~,7). To determine the relative inhibition of catecholamine-stimulated lipolysis, 20 ~i of viprostol in ethanol was added to the incubations in triplicate to achieve a final concentration of 10-3 to 10-7M, and in the presence of epinephrine. For the determination of the comparative antilipolytic activity, either viprostol or a naturally-occurring prostaglandin (PGEI, PGE. or PGF. ; Ono Pharmaceutical Co., Osaka, Japan) was added £o the a~pocyte incubation in the presence of epinephrine. Each was dissolved in absolute ethanol, and added in a volume of 20 ~i at a final concentration of 10 ~M. Preliminary experiments indicated that the ethanol vehicle alone did not affect the results. Following the assessment of total medium glycerol (8), fat cell size and number were calculated (9), ultimately allowing the expression of lipolysis per adipocyte. The relative capability of each PG to inhibit catecholamine-stimulated lipolysis was calculated by comparing the absolute glycerol in PG-containing vials to those containing only the catecholamine (100% lipolysis). Values from each group were expressed as the mean ± the standard error of the mean (SEM), and were considered significantly different at p<.05. Linear regression analysis was used to determine the effective concentration at which 50% of the hormone-stimulated lipolysis wouldbe inhibited by viprostol (10). Mean values were compared using the t-test.
768
MAY 1987 VOL. 33 NO. 5
PROSTAGLANDINS
RESULTS Viprostol exhibited a dose-dependent inhibition of catecholamine-stimulated lipolysis (Table i). In isolated adipocytes, maximally stimulating concentrations of epinephrine (i0 M) alone resulted in over a 30x increase above basal lipolysis, viprostol was ineffective in the inhibition of hormone-stimulated lipolysis ~t 10-6 and 10-TM. A reduction in lipolysis was observed at i0- M, but significant inhibition in glycerol release was observed only at 10-3 and 10-4M. The inhibition of 50% of the observed catecholamine-stimulated lipolysis was estimated as 3.45x10-4M. -5
,
Table i. Concentration of Viprostol
(M) 10-3
.
Effect of Viprostol on CatecholamineStimulated Lipolysis Glycerol Release (~mol/106fc/hr) .354±.04**
i0 -4
1.16 ±.21"
10-5
2.07 ±.39
10-6
2.55 ±.35
10-7
2.59 ±.39
Basal Epinephrinestimulated
Adipocyte Characteristics Diameter: 75.4±2.6 microns Volume: 260±26 picoliters Number per flask: .436±.051 x 106
Rat body weight: 504±27 g
.071±.018 2.45 ±.36
Values are mean±SEM of 7 separate experiments with triplicate incubations for each concentration. * Significantly different from epinephrine-stimulated value at p<.05 and **p<.001. In a separate set of experiments, the comparative antilipolytic activity of viprostol and naturally-occurring PG's was assessed. As seen in Figure i, at equimolar concentrations (i0 ~M), PGE I and PGE~ inhibited lipolysis by approximately 65%, where catecholamine-s%imulated glycerol release was considered as 100% lipolysis. PGF2~ was the least potent of the naturally-
MAY 1987 VOL. 33 NO. 5
769
PROSTAGLANDINS
occurring PG's, while viprostol inhibited maximally induced lipolysis by approximately 37%. When tested individually, either viprostol or PGF2. was capable of significantly (p<.05) reducing epinephrine-stimulated glycerol release. However, the antilipolytic potency of PGEI or PGE2 was significantly greater than that of either viprostol or PGF2~, based upon a significant reduction (p<.025) of released ~lycerol in the presence of equimolar concentrations of either PGEI or PGE 2 compared directly to PGF. or viprostol. From these experiments, it is estimated that vlprostol exhibited approximately 50% of the activity of PGE in vitro. Figure I:
Comparative Effect of Viprostol (CL 115,347) in the Inhibition of CatecholamineStimulated Lipolysis
70
q_
60
50 r--1 40
PGE]
HIll]I]]] PGE 2
Relative Inhibition of L i p o l y s i s (%)
PGF2~
30
m
ll-
r~.-..-..-..'~ CL 115, 347
M_n..:'
20
10
0
Because of the maximally-stimulating concentrations of catecholamine used in these experiments, incubations including a methyl xanthine provided insight toward the integrity of the ~-adrenceptor. As shown in Table 2, IBMX significantly stimulated lipolysis, which was dramatically and significantly inhibited by 10 ~M viprostol. Since lipolysis due to IBMX remained intact, these data imply that viprostol acts distally to the ~-adrenoceptor.
770
MAY 1987 VOL. 33 NO. 5
PROSTAGLANDINS
Table 2.
Effect of Isobutyl-methyl-xanthine (IBMX) on Viprostol's Antilipolytic Activity
Incubation Condition
Glycerol Release (pmol/106 fc/hr)
Adipocyte Characteristics
Basal
1.20±.18
Diameter:
Basal + Viprostol
1.90±.23
108.4±3.4 microns
(i00 ~M) Epinephrine
Volume: 4.62±.66a
779±71 picoliters
Epinephrine + Viprostol
1.66±.08
Number per flask:
IBMX
8.35±1.55"
(i0 ~M)
.259±.034 x 106
(1 mM) IBMX + Viprostol
Rat Body Weight: 1.79±.25
722±46 g
Values are mean±SEM of 2-5 separate experiments, with each incubation condition in triplicate. No significant differences were apparent between Basal and Basal + Viprostol. a Significantly different from Basal-Viprostol value at p<.025. DISCUSSION These studies indicate that viprostol exhibits antilipolytic activity in vitro in a dose-dependent manner, but that the inhibition of lipolys-is was evident only at relatively high concentrations of the compound. In fact, other investigators have estimated the fiftypercent inhibition of catecholamine stimulated lipolysis for PGE 2 in the micromolar range, indicating an approximate i00 times greater potency than viprostol (3). We have usedmaximally stimulating concentrations of catecholamine, however, and the aforementioned report of relative potency of PG used a submaximal concentration of catecholamine. Importantly, the dose-response for isolated adipocyte release of glycerol following catecholamine stimulation is quite variable at sub-maximal concentrations (ii), and maximal stimulation has often been considered necessary when assessing lipolysis in isolated fat cells (6,11). The activity of viprostol in terms of the relative inhibition of lipolysis should therefore also be analyzed in comparison to the antilipolytic activity of naturally-occurring PG. The relative inhibition that we observed agrees well with published data using similar incubation conditions (i). From these data, it
MAY 1987 VOL. 33 NO. 5
771
PROSTAGLANDINS
appears that viprostol is approximately 50% as potent as PGE, and equally potent to PGF 2 . The antilipolytic potency of viprostol may also have been underestimated because, as indicated by the structural formula, viprostol is a mixture of multiple molecules. Preliminary observations in our laboratory indicate that the separated 1-isomer is approximately 4x more potent that viprostol with respect to antihypertensive activity. In summary, viprostol is a synthetic PGE_ analog in which structural alteratlons have resulted in a reductlon in antilipolytic activity when compared directly to the naturally-occurring hormone. While viprostol exhibits antilipolytic activity, the concentrations required to inhibit maximally-induced lipolysis in vitro are indicative of a diminution in antilipolytic activ-lty--~lowing movement of the hydroxyl group to the 16-carbon position. Interestingly, this compound is documented to produce other biological effects associated with PGE, and after a single oral or transdermal administration (4,12). In a similar manner, future experiments examining antilipolytic activity in vivo may therefore be used to better assess the effects of vipro-sto~n the regulation of lipid metabolism. REFERENCES i.
Steinberg, D., M. Vaughan, P. Nestel, O. Strand, and S. Bergstrom. Effects of the prostaglandins on hormoneinduced mobilization of free fatty acids. J. Clin. Invest. 43: 1533, 1964.
2.
Robertson, R.P., and S. Little. Down-regulation of prostaglandin E receptors and homologous desensitization in isolated adipocytes. Endocrinology 113: 1732, 1983.
3.
Axelrod, L., Trzepacz, P., Zusman, R., and Martin, D. Antilipolytic effect of prostaglandin E 2 analogues: therapeutic implications. Life Sci. 18: 627, 1976.
4.
Chan, P.S., P. Cervoni, M. Ronsberg, R. Accomando, G. Quirk, P. Scully, and L. Lipchuck. Antihypertensive activity of d,l-15-deoxy-16-hydroxy-16(~/8)-vinyl prostaglandin E 2 methyl ester (CL 115,347), a new orally and transdermally longacting antihypertensive agent. J. Pharmacol. Exp. Ther. 226: 726, 1983.
5.
Rodbell, M. Metabolism of isolated fat cells. Chem. 239: 375, 1964.
6.
Crandall, D.L., S. Fried, A. Francendese, M. Nickel, and M. DiGirolamo. Lactate release from isolated adipocytes: influence of cell size, glucose concentration, insulin and epinephrine. Horm. Metab. Res. 15: 326, 1983.
772
J. Biol.
MAY 1987 VOL. 33 NO. 5
PROSTAGLANDINS
7.
Birnbaum, J.E., P. Cervoni, P.S. Chan, S. Chen, M. Floyd, C. Grudzinskas, M. Weiss, and F. Dessy. Prostaglandins and congeners 29. (16RS)-±-15-deoxy-16-hydroxy-16-vinylprostaglandin E 2 , an orally and transdermally active hypotensive agent of prolonged duration. J. Med. Chem. 25: 492-4, 1982.
8.
Pinter, J.D., J. Hayashi, and J.A. Watson. Enzymatic assay of glycerol, dihydroxyacetone and glyceraldehyde. Arch. Biochem. Biophys. 121: 404, 1967.
9.
DiGirolamo, M., S. Mendlinger, and J. Fertig. A simple method to determine fat cell size and number in four ma~nalia species. Am. J. Physiol. 221: 850, 1971.
i0. Snedecor, G., and W. Cochran. Statistical Methods, 6th Ed. Iowa State Univ. Press, Ames, 1967. p. 549. ii. Zinder, O., and B. Shapiro. Effect of cell size on epinephrine- and ACTH-induced fatty acid release from isolated fat cells. J. Lipid Res. 12: 91-95, 1971. 12. Crandall, D.L., B.M. Goldstein, R. Gabel, F.H. Lizzo, and P. Cervoni. Effects of the antihypertensive prostaglandin analog CL 115,347 on cardiac output distribution in the spontaneously hypertensive rat. J. Cardiovasc. Pharmacol. 7: 996, 1985.
Editor: R.P. Robertson
MAY 1987 VOL. 33 NO. 5
Received:
9-17-86
Accepted:
3-30-87
773