Medical contraindications and issues for consideration in the use of once-a-month injectable contraceptives.

A number of once-a-month injectable contraceptives have been studied over the last 30 years and at least three different formulations are currently in use worldwide. Each of these formulations contains an ester of the natural estrogen, estradiol, and a synthetic progestin. This is in contrast to the combined oral contraceptives which contain synthetic analogs of both estradiol and progesterone. This paper considers medical contraindications or precautions related to the use of once-a-month injectable formulations and suggests some considerations for their programmatic use, particularly in the developing world. Because once-a-month injectables contain both an estrogen and a progestin, the precautions related to their use are similar to those of combined oral contraceptives; however, since the estrogen component is a natural estrogen, administration is parenteral and the circulating levels reach peaks that are in the range of those of the normal preovulatory phase of the menstrual cycle, some of these precautions may be very conservative. Absolute contraindications to the use of monthly injectables are current or suspected pregnancy, and estrogen-responsive tumors of the breast or genital tract. Other factors have been termed precautions for use rather than absolute or relative contraindications. Primary precautions are conditions under which a woman should probably not use monthly injectables, but if she does, she should be monitored closely. These include current or individual history of cardiovascular disease, current liver disease or liver tumors, and over age 40 if a smoker. Secondary precautions to use are those which should be considered very carefully and balanced against the risks of not using the particular method. These include suspicion of breast or genital tract cancer until cancer is ruled out, unexplained abnormal vaginal bleeding, use of drugs which might affect metabolism and decrease effective levels of circulating steroids, breastfeeding, and having combinations of several risk factors for cardiovascular disease. These precautions are categorized as proposed, based on the fact that most women in the developing world face a risk from pregnancy that is probably far greater than the risks from any of the present formulations of steroidal contraceptives. A key consideration during programmatic use of monthly injectables is attention to compliance with follow-up schedules to minimize the risk of pregnancy. Because little information on monthly injectables is available that is directly related to these precautions, additional research studies will be required in order to reconsider whether some of the listed precautions are valid or others should be considered.

PIP: Researchers have studied various once-a-month injectable contraceptives for the last 30 years. Women are currently using the formulations Cyclofem, Chinese Injectable No. 1, and Deladroxate. Mesigyna is scheduled to be introduced in 1994. All these once-a-month injectables contain a synthetic progestin and an ester of the natural estrogen, estradiol. Combined oral contraceptives (OCs) contain synthetic analogs of estradiol and progesterone. Very limited direct research on contraindications to monthly injectable contraceptives exist. In addition to a review of this limited research, an examination of OCs, postmenopausal hormone replacement therapies which use natural estrogens, and progestin-only contraceptives may identify possible contraindications. Absolute contraindications to use of once-a-month injectables include current or suspected pregnancy and estrogen-dependent tumors of the breast or genital tract. A woman with the following primary precautions for use should probably not use monthly injectables: a current or individual history of cardiovascular disease, current liver disease or liver tumors, or age over 40 if a smoker. If she does use them, however, providers should closely monitor her. The client and provider should carefully consider secondary precautions for use and balance them against the risks of not using another method. The secondary precautions are suspected breast or genital tract cancer, unexplained vaginal bleeding, use of drugs which might interact with the steroids in the injectables and decrease the effectiveness of circulating steroids, breast feeding, and combinations of several risk factors for cardiovascular disease. Most women in developing countries are likely to face a much higher risk from pregnancy than from current hormonal formulations. Compliance with follow-up schedules is needed to reduce the risk of pregnancy. More research is needed to determine whether the proposed precautions are valid or whether others should be included.

Adenosine inhibits prolactin and growth hormone secretion in a clonal pituitary cell line.

Although purine nucleosides have been shown to regulate the secretion of several peptide and steroid hormones, effects on pituitary hormone release have not been reported. We show here that in the clonal GH4C1 pituitary cell line maximal concentrations of adenosine (greater than or equal to 50 microM) inhibited PRL and GH secretion by 40%. Adenosine deaminase abolished the inhibitory effect of adenosine but not that of SRIF or (-)N6(R-2-phenylisopropyl)adenosine (PIA), a nonhydrolyzable adenosine analog. Furthermore, this enzyme increased basal secretion by 50%, and analysis of the incubation medium by HPLC demonstrated that the cells secreted biologically effective concentrations of adenosine. These results indicate that adenosine produced in culture tonically inhibits hormone release. In other target cells, adenosine inhibition is mediated by two types of binding sites: an extracellular Ri-site requiring an intact ribose moiety or an intracellular P-site requiring an intact purine ring. Four lines of evidence indicate that in GH4C1 cells, adenosine acts at an Ri-site. PIA, an Ri-site-specific agonist, was a potent inhibitor of hormone release (ED50 = 30 nM). Theophylline, an Ri-site antagonist, competitively inhibited the action of PIA (Ki = 2.4 microM). 3) 2'5'-Dideoxyadenosine, a P-site-specific agonist, did not inhibit PRL release even at a concentration of 1 mM. 4) Dipyridamole, an adenosine uptake inhibitor, did not reduce adenosine inhibition. In addition to its effect on basal secretion, PIA inhibited stimulation of hormone release by vasoactive intestinal peptide and TRH. PIA also reduced vasoactive intestinal peptide-stimulated cAMP accumulation by 75%, consistent with its action to inhibit adenylate cyclase via Ri receptors in other targets. Since PIA inhibition of PRL release and cAMP accumulation was not additive with the effects of SRIF and carbamyl choline, these inhibitors may act via a common rate-limiting step. Our results demonstrate that adenosine activates an Ri-type of adenosine receptor in GH4C1 cells and that the production of adenosine under normal culture conditions causes autocrine inhibition of secretion.

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium.

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.

Relative potency of progestins used in oral contraceptives.

Epidemiologic studies indicate that the risk of complications with oral contraceptive use is related to the steroid content and potency of the various formulations. This paper summarizes human data in which potencies of progestins in oral contraceptives can be compared. Data on delay of menses and endometrial subnuclear vacuolization, an indirect assessment of glycogen deposition, are presented. The relative effects of various progestins on serum lipids and lipoproteins are also summarized. The object of this review is to examine the available scientific evidence which generally supports the conclusion that there is a marked similarity of potency of the dose of various progestins used in many of the formulations currently available in the U.S. The progestins norethindrone, norethindrone acetate and ethynodiol diacetate are roughly equivalent in potency while norgestrel is roughly five to ten times and levonorgestrel ten to 20 times as potent.

Mechanisms of somatostatin action in pituitary cells.

We have examined the mechanisms by which S-14 inhibits pituitary hormone secretion in a homogeneous cell population: the clonal GH4C1 cell line. The S-14 receptor in GH4C1 cells is coupled to Ni, a guanine nucleotide binding protein which mediates S-14-induced inhibition of VIP-stimulated adenylate cyclase activity, cyclic AMP production and hormone secretion. In addition, a functional Ni is required for S-14 to inhibit basal hormone secretion, an action which appears to be independent of cyclic AMP concentrations. Accumulating evidence indicates that the mechanism of S-14 action in somatotrophs is similar to that in GH4C1 cells. Although S-14 consistently inhibits basal GH secretion, its effects on basal cyclic AMP levels in normal pituitary cells are variable and often not significant (10-14). In contrast, S-14 inhibits prostaglandin and growth hormone releasing factor (GRF) stimulated cyclic AMP accumulation and GH release in parallel. Furthermore, S-14 partially blocks prostaglandin and GRF stimulation of adenylate cyclase activity in rat anterior pituitary membranes. Finally, pretreatment of primary cultures of rat pituitary cells with IAP antagonizes S-14 inhibition of both basal and GRF-stimulated GH release.

Prostaglandin F2 alpha inhibits luteinizing hormone (LH)-induced increase in LH receptor binding to isolated rat luteal cells.

The effect of the luteolytic hormone prostaglandin F2 alpha (PGF2 alpha) on parameters of LH receptor binding to isolated rat luteal cells was examined. The equilibrium binding constant (0.55 X 10(10) M-1), the association rate constant (0.89 X 10(8) M min-1), and the dissociation rate constant (1.70 X 10(-2) M min-1) were not significantly altered by PGF2 alpha. However, as [125I]iodohCG binding approached equilibrium (less than 2 h) and at equilibrium (greater than 3 h), PGF2 alpha, but not PGE2, consistently reduced LH binding by 10-20%. The LH receptor-binding capacity, determined by Scatchard analysis of [125I]iodo-hCG or [125I]iodo-hLH binding, was reduced from 7.5 +/- 0.1 to 6.4 +/- 0.1 X 10(4) receptors/cell in the presence of PGF2 alpha. This reduction of total cell-bound radioactivity by PGF2 alpha was not due to a change in the rate of hormone internalization and degradation. However, when cells were briefly treated with a pulse of LH (5 ng/ml; 5-10 min), [125I] iodo-hCG (LH) binding increased 20-30% within 2 h, and PGF2 alpha prevented this LH-induced increase. A similar pulse of cAMP analogs did not alter LH binding. We conclude that while the initial binding of LH to its receptor is not altered by PGF2 alpha, it does reduce the final equilibrium level of LH binding by a mechanism that involves a block in the appearance of LH-induced cryptic receptors.

Calcium is an inhibitor of luteinizing hormone-sensitive adenylate cyclase in the luteal cell.

Both prostaglandin F2 alpha (PGF2 alpha) and LHRH inhibit LH-stimulated cAMP accumulation and progesterone secretion in the intact luteal cell, but have no effect on LH-sensitive adenylate cyclase activity in isolated membranes. The present studies were conducted to assess the possibility that calcium (Ca2+) may mediate the inhibitory activity of PGF2 alpha and LHRH in the rat luteal cell. Removal of extracellular Ca2+ significantly enhanced cAMP accumulation in response to LH by about 2-fold, but blunted LH-stimulated progesterone secretion. Incubation of luteal cells with A23187 caused a highly significant and dose-related decrease in LH-stimulated cAMP accumulation with a concentration for half-maximal inhibition (IC50) of about 1 microM. No effect of A23187 was seen on LH-sensitive adenylate cyclase activity, but the ionophore elicited significant inhibition of LH-stimulated intracellular cAMP accumulation in the presence of isobutyl-methylxanthine (MIX), a phosphodiesterase inhibitor. Inhibition by A23187 was Ca2+ dependent, since a decrease in extracellular Ca2+ to less than 100 microM completely blocked the effect of the ionophore. A23187 also significantly inhibited LH-stimulated progesterone secretion in response to LH or cholera toxin and inhibited cholera toxin-stimulated cAMP accumulation in the absence or presence of MIX. In incubations of isolated luteal membranes, Ca2+ produced a dose-dependent inhibition of LH-stimulated adenylate cyclase activity in the absence or presence of MIX at free Ca2+ levels between 5-20 microM (IC50, approximately 10 microM). Depletion of extracellular Ca2+ had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha in the intact cell, and the inhibitory activity of LHRH was slightly reduced, but not abolished, by depletion of extracellular Ca2+. Verapamil, a Ca2+ channel blocker, had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha or LHRH. It is concluded that an acute increase in intracellular Ca2+ inhibits activation of adenylate cyclase by LH in the rat luteal cell. This conclusion is based on studies that showed enhanced cAMP accumulation by LH in Ca2+-depleted media, Ca2+-dependent inhibition of LH-stimulated cAMP production by a Ca2+ ionophore, and direct inhibition of LH-sensitive adenylate cyclase activity by Ca2+ in luteal membranes. It is suggested that a similar effect occurs in response to PGF2 alpha or LHRH in the luteal cell, but inhibition by these luteolytic agents is not dependent on an influx of extracellular Ca2+, but, rather, is due to an increase in intracellular Ca2+ by other mechanisms.

Inhibitory characteristics of prostaglandin F2 alpha in the rat luteal cell.

Enzymatically dispersed and enriched preparations of rat luteal cells were used to characterize the antigonadotropic effects of prostaglandin (PG) F2 alpha. The half-maximal dose (ED50) of LH for stimulation of cAMP accumulation and progesterone secretion was 100 and 25 ng/ml, respectively. Methylisobutylxanthine (MIX) had no effect on the ED50 of LH on cAMP accumulation but reduced the ED50 of LH on progesterone secretion from 25 to 10 ng/ml. PGF2 alpha inhibited the tropic responses to LH by 55-70% within minutes at concentrations of PGF2 alpha within the physiological range. For example, 2-4 nM PGF2 alpha inhibited LH-stimulated cAMP accumulation by 50% (IC50). PGF2 alpha reduced the maximum cAMP response to LH but had no effect on the ED50 of LH for cAMP accumulation whereas PGF2 alpha increased the ED50 of LH on progesterone secretion by 5-7-fold. Inhibition by PGF2 alpha appeared to be unrelated to an effect on cAMP phosphodiesterase activity or to changes in parameters of LH receptor binding activity. No inhibition by PGF2 alpha was evident on LH-stimulated cAMP accumulation in isolated membranes. PGF2 alpha had little effect on cAMP accumulation in response to cholera toxin or forskolin but produced significant inhibition of progesterone secretion in response to cholera toxin or dibutyryl cAMP [Bu)2cAMP). It is concluded that the antigonadotropic effect of PGF2 alpha in the luteal cell is due to two interrelated actions: inhibition of activation of cAMP accumulation by LH and inhibition of the luteal cell response to cAMP. Since PGF2 alpha had no effect in the broken cells, it is suggested that the action of PGF2 alpha may be mediated by a second messenger in the intact cell.

Somatostatin inhibits basal and vasoactive intestinal peptide-stimulated hormone release by different mechanisms in GH pituitary cells.

Somatostatin (SRIF) inhibits both basal and vasoactive intestinal peptide (VIP)-stimulated hormone secretion by the GH4C1 clonal strain of rat pituitary tumor cells. We have previously shown that SRIF inhibits cAMP accumulation stimulated by VIP but does not alter basal cAMP levels in this cell line. To determine the importance of changes in cAMP accumulation in the mechanism of SRIF action, we have compared the effect of SRIF on hormone release stimulated by VIP and two other secretagogues which increase effective intracellular cAMP concentrations: forskolin and 8-Bromo-cAMP (8-Br-cAMP). VIP stimulated GH and PRL secretion to the same maximal extent (220% of control) with similar ED50 values (0.37 +/- 0.03 and 0.43 +/- 0.08 nM, mean +/- SE, respectively). SRIF (100 nM) reduced maximal VIP-stimulation of both GH and PRL release from 220 to 140% of control; however, it did not significantly change the ED50 values for VIP. The effect of SRIF on VIP-stimulated hormone release parallels its action on VIP-stimulated cAMP accumulation. Furthermore, the concentrations of SRIF required to produce half-maximal inhibition of VIP-stimulated GH and PRL release (0.8 +/- 0.2 nM and 0.7 +/- 0.1 nM, respectively) were similar to its potency to inhibit VIP-stimulated cAMP accumulation (1.2 +/- 0.1 nM). These data indicate that changes in cAMP levels mediate inhibition of VIP-stimulated hormone secretion by SRIF. Forskolin increased cAMP accumulation with an ED50 value of 2.4 +/- 0.5 microM. A maximal concentration of forskolin (100 microM) stimulated cAMP accumulation to a greater extent than 100 nM VIP (34 +/- 4-fold vs. 9 +/- 1-fold). Together, forskolin (100 microM) and VIP (100 nM) stimulated cAMP accumulation by more than 50-fold. However, PRL secretion in response to maximal concentrations of VIP or forskolin individually or together were the same (approximately 200% of control). These results support the conclusion that both compounds stimulate PRL secretion by a cAMP-mediated mechanism which can be fully activated by either one alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Somatostatin inhibits vasoactive intestinal peptide-stimulated cyclic adenosine monophosphate accumulation in GH pituitary cells.

Somatostatin (SRIF) has previously been shown to inhibit both basal and hormone-stimulated PRL secretion from GH4C1 cells, a clonal strain of rat pituitary tumor cells. In this study we examined the ability of SRIF to modulate cAMP accumulation in GH4C1 cells to determine whether such alterations mediate its biological effects. SRIF did not cause statistically significant changes in basal cAMP accumulation. Of six PRL secretagogues examined, only vasoactive intestinal peptide (VIP) increased cAMP accumulation significantly: TRH, bombesin, epidermal growth factor, insulin, and the tumor promoter, phorbol-12,13-dibutyrate were without effect. When SRIF was added simultaneously with VIP, it inhibited maximal VIP-stimulated cAMP accumulation (55 +/- 3%, mean +/- SE) without changing the ED50 for VIP (3.0 +/- 0.2 nM). Inhibition by SRIF was not due to altered kinetics of VIP stimulation, since the half-time for VIP-stimulated cAMP accumulation was 2 min both in the absence and presence of 100 nM SRIF. SRIF did not inhibit isobutylmethylxanthine-stimulated cAMP accumulation, and the presence of 0-10 mM isobutylmethylxanthine did not alter the inhibitory effect of SRIF on VIP-stimulated cAMP accumulation. Therefore, SRIF must act primarily to modulate VIP activation of adenylate cyclase activity. Inhibition of VIP-stimulated cAMP accumulation occurred at concentrations of SRIF (ID50 = 1.2 +/- 0.1 nM) close to the equilibrium dissociation constant for receptor binding (Kd = 0.6 +/- 0.2 nM). Furthermore, the potencies of a series of SRIF analogs to inhibit VIP-stimulated cAMP accumulation correlated with the apparent Kd of each peptide for binding to the SRIF receptor. In addition, SRIF did not reduce VIP-stimulated cAMP accumulation in GH(1)2C1 cells, which lack SRIF receptors. We conclude that SRIF inhibits VIP-stimulated cAMP accumulation by a receptor-mediated process that may be causally related to the ability of SRIF to inhibit VIP-dependent PRL secretion.

Mechanisms of PGF2 alpha action in functional luteolysis.

The luteolytic action of PGF2 alpha appears to be due to loss of gonadotropin support of corpus luteum function. This "chemical hypophysectomy" produced by PGF2 alpha in the rat has several components: 1. A rapid block of gonadotropin uptake in vivo by an unexplained mechanism. 2. A rapid and direct block of adenylate cyclase activation by the LH receptor complex in isolated luteal cells. 3. An eventual loss of LH receptor binding activity produced by inhibition of prolactin action.

Mechanism of the rapid antigonadotropic action of prostaglandins in cultured luteal cells.

A reproducible method for dissociation and culture of rat luteal cells is described. The concentration of LH required to produce half-maximal stimulation of progesterone secretion was 50 ng/ml. The effects of prostaglandin E(2) (PGE(2)) and prostaglandin F(2alpha) (PGF(2alpha)) on basal and luteinizing hormone (LH)-stimulated progesterone production were examined. Both prostaglandins stimulated basal progesterone production but PGE(2) was about twice as active, showing a 2-fold maximal stimulation at 0.75 muM. When either prostaglandin was incubated simultaneously with LH, a dose-dependent inhibition of progesterone secretion occurred; PGF(2alpha) was 4 times more active than PGE(2), showing 50% inhibition at a concentration of 40 x nM. Thus, both prostaglandins are more active as antagonists than as agonists of LH with respect to progesterone secretion. PGF(2alpha) also inhibited LH-stimulated adenylate cyclase activity and cyclic AMP accumulation. The block in progesterone secretion was reversed by addition of dibutyryl cyclic AMP (1 mM) but not by theophylline (5 mM) alone. These data and the finding that PGF(2alpha) did not affect the specific binding activity of the LH receptor in intact luteal cells indicate that the rapid action of prostaglandins in luteal cells is due to a block of LH-dependent production of cyclic AMP which results in a decrease in progesterone secretion.