Effects of treatment of normal and hyperprolactinemic rats with cyclosporine in vivo on the release of gonadotropins and prolactin from their pituitaries in vitro.

Cyclosporine (CyA) is extremely useful as an immunosuppressant and it is believed that at least some of its actions are due to antagonizing PRL effects. To determine whether the reported ability of CyA to inhibit gonadotropin release can be modified by PRL, we have examined the effects of treatment of normal and hyperprolactinemic rats with CyA in vivo on the release of LH, FSH and PRL from their pituitaries in vitro. Hyperprolactinemia was induced by implantation of capsules containing diethylstilbestrol (DES) and the animals were examined while the capsules were still in place (DES-IN) or after they had been removed (DES-OUT). Treatment with CyA significantly reduced plasma LH levels in control DES-IN rats without reducing basal LH release from the pituitaries of these animals in vitro. In the DES-IN rats, CyA exposure in vivo did not modify plasma PRL levels, but reduced PRL release in vitro, and interfered with the inhibitory action of dopamine (DA) on PRL release. The effect of DA on gonadotropin release in vitro was modified by CyA treatment. Administration of CyA failed to antagonize the suppressive effects of hyperprolactinemia on plasma LH and FSH levels or on the basal rates of gonadotropin release by incubated pituitaries. We conclude that CyA can reduce PRL release but does not interfere with the actions of PRL on anterior pituitary function.     

Prolactin and growth hormone in the regulation of the immune system

Evidence implicating prolactin (PRL) and growth hormone (GH) in the regulation of the immune system has been reviewed. Hypophysectomized animals have deficiencies in both cell-mediated and humoral immunological functions and either PRL or GH corrects these deficiencies. Animals administered bromocryptine, a drug that specifically blocks PRL release, have impaired immune responses similar to hypophysectomized animals, and again both PRL and GH correct these deficiencies. Genetically dwarf animals, which lack both PRL and GH, are also immunocompromised, and once again PRL and GH can correct the deficiencies. In dwarf animals, however, fewer studies have examined PRL actions. In growth-deficient children, immune function is not dramatically altered and basal secretion of GH has been reported. Very few clinical studies have examined whether PRL secretion is also deficient, and this may explain why a clear loss in immune function is not evident in growth-deficient children. In a number of species, including man, both PRL and GH stimulate thymic function and increase the secretion of thymulin, a thymic hormone. No studies, however, have reported on the effects of PRL and GH on other thymic hormones. A number of studies have reported in vitro effects of PRL and GH on cells involved with immunity, and the presence of high-affinity PRL and GH receptors have been observed on a number of these cells. The action of GH on the proliferative response of cells involved with immunity in vitro appears to be mediated by the production of insulin-like growth factor I. The effect of PRL on insulin-like growth factor I production by these cells has not been examined. One of the most consistent findings from in vitro studies is that prolactin antisera blocked a number of immune reactions. This led to the discovery that cells involved with immunity appear capable of producing PRL and GH, but the physiological significance of these observations have not been explored. There is a great need to identify the cell types responding to PRL and GH and this should be a goal of future investigations. There is also a need for investigators to be aware that both PRL and GH are involved in the regulation of the immune system and to design experiments to elucidate where each functions in the maturation cascade of cells involved with immunity. From the evidence available, it is apparent that PRL and GH have an important function in the immune system and future investigations should be directed toward elucidating their site(s) of action.     

In vitro release of prolactin by thyrotropin-releasing hormone: influence of dopamine, thyroxine, and cycloheximide.

An acute incubation procedure, using explanted normal rat hemipituitaries pretreated with fresh plasma obtained from pituitary donor animals, was employed to further investigate the in vitro stimulation of prolactin (PRL release by thyrotropin-releasing hormone (TRH). Pretreatment with dopamine (0.1 microgram/ml) caused a 30-50% decrease in the amount of PRL released into incubation media; the inhibitory effect of dopamine was not reversed by treatment with 0.5-6.0 ng. TRH, although these TRH concentrations consistently stimulated PRL release from pituitaries not exposed to dopamine. Treatment with thyroxine (10(-6) to 10(-5) M) showed a competitive inhibition of thyrotropin release by TRH (0.5 ng), but was without effect on TRH-stimulated PRL release. Cycloheximide (100 microgram/ml) blocked a net increase in PRL levels. TRH, nevertheless, significantly increased PRL release in the presence of cycloheximide. The results indicate that neither dopamine nor thyroxine compete with TRH in causing PRL release, and that the TRH stimulation of PRL release is unrelated to ongoing levels of hormone synthesis.     

Generation of hypophysiotropic activity in hypothalamic extracts in vitro.

The luteinizing hormone (LH) and follicle stimulating hormone (FSH) releasing activity, as well as the prolactin (PRL) release-inhibiting activity were measured in both neutral aqueous, and acid ethanolic extracts of rat hypothalami. LH and FSH-releasing activities were detectable only in the latter type of extract, whereas PRL release-inhibiting activity appeared in both. Neutral ultrafiltrates of the neutral extracts contained no gonadotropin releasing activity, however, acidification of the filtration medium induced its appearance. PRL release was inhibited by both neutral and acid filtrates. These results suggest that LH and FSH releasing factor(s) may be stored in the hypothalamus in an inactive form from which the active peptide is generated in vitro under acid conditions; however, this does not appear to be true for the component(s) responsible for the inhibition of PRL release.     

The effect of clozapine on prolactin secretion at the level of the lactotroph

Clozapine is an antipsychotic drug which is unusual in that it has no dopamine receptor-blocking activity. Previous studies gave conflicting results whether administration of clozapine induces hyperprolactinemia. In the present study it was shown that a wide concentration range of clozapine does not interfere with dopamine-mediated inhibition of prolactin (PRL) secretion by normal cultured rat pituitary cells. This in contrast to other neuroleptics, like haloperidol and trifluoperazine. Clozapine does also not antagonize norepinephrine-mediated inhibition of PRL secretion. Clozapine exerts at micromolar concentrations a direct inhibitory action on PRL release by cultured normal rat pituitary cells. In cultured rat pituitary tumor cells, these high concentrations of clozapine directly inhibit PRL release as well as the DNA content of the cells, suggesting a direct antimitotic action. In this model clozapine was about 5-10 times less potent than trifluperazine. Clozapine and trifluoperazine exert an additive inhibitory action both on PRL release and on the DNA content of the pituitary tumor cells. It is concluded that clozapine does not interfere at the pituitary level with dopamine-mediated inhibition of PRL release. At micromolar concentrations clozapine may act on lactotrophs as a calmodulin-inhibitor. These observations suggest that the transient PRL-releasing effects which have been observed in both animal and human studies after clozapine administration are mediated via supra-pituitary actions of the drug.     

A comparison of the effects of suckling or transient dopamine antagonism on thyrotropin-releasing hormone and suckling induced prolactin release in lactating rats

Prolactin (PRL) release was studied in mid-lactational female rats by comparing the stimulatory influence of suckling to a drug protocol that mimics the effect of suckling on the anterior pituitary (AP). Animals that nursed pups for 15 minutes and were allowed to suckle again 60 minutes later for 10 minutes, released PRL effectively during both nursing episodes; however, in animals that received the dopamine (DA) agonist 2-Br-alpha-ergocryptine maleate (CB-154, 0.5 mg/rat i.v.) at the end of the first nursing period did not show an increase in plasma PRL to a second suckling stimulation by the pups. When thyrotropin releasing hormone (TRH) was substituted for the second suckling period in CB-154 treated rats, a slight increase in plasma PRL occurred 5 minutes after the injection. In a third study we transiently blocked the action of DA at the AP by injecting the DA antagonist domperidone (0.01 mg/rat i.v.), followed 5 minutes later by the administration of CB-154. One hour later animals were either allowed to suckle pups for 10 minutes or were injected with TRH. Treatment with TRH resulted in an 11 fold increase in plasma PRL but suckling was completely ineffective in inducing PRL release. These data suggest that the lack of PRL release to suckling in CB-154 treated rats was due to inhibitory effects of CB-154 on neural mechanisms which link nursing to PRL release. In addition, the data show that pharmacologic DA antagonism affects TRH releasable PRL more than does suckling. This may be due to a reduction, by suckling, of the pool of PRL that is available to be released by TRH administration.     

Does androgen influence prolactin secretion?

In both intact and castrated male and female rats, administration of the A-ring reduced androgen, dihydrotestosterone (DHT), consistently failed to stimulate prolactin (PRL) secretion although it inhibited LH release and, in males, stimulated ventral prostate growth. In intact females, but not in the other types of rat, DHT actually suppressed PRL release. These findings do not support generalizations, based entirely on findings with testosterone, that both "androgens" and estrogens exert stimulatory actions on PRL secretion. The distinct stimulatory effects of testosterone and its esters on PRL secretion seem attributable, not to their androgenic actions per se, but to the ability of testosterone to form estrogenic metabolites. This ability does not appear to be shared by the "pure" androgen, DHT.     

The met-enkephalin analog FK 33-824 directly inhibits ACTH release by the rat pituitary gland in vitro

Systematic administration of the enkephalin analog FK 33-824 was previously shown to stimulate PRL secretion and to inhibit ACTH secretion in man. Naloxone prevented the effect on PRL release, but not on ACTH release. In this study, the direct action of this analog on hormone release by rat anterior pituitary lobes $\text{in}$$\text{vitro}$ were investigated. 1 uM FK 33-824 inhibited basal ACTH secretion by anterior pituitary glands in vitro, while 0.1 uM and 1 uM attenuated the lysine vasopressin stimulated ACTH release. Naloxone did not reverse the inhibitory action of the analog on ACTH release. β-Endorphin (0.01 - 1 uM) did not directly affect ACTH release. Basal and dopamine-induced inhibition of PRL release by anterior pituitary glands was neither influenced by FK 33-824 (0.1 and 1 uM), nor by β-endorphin (0.1 and 1 uM) with or without bacitracin. This study shows that the long-acting met-enkephalin analog FK 33-824 differentially affects PRL and ACTH secretion by the pituitary gland. It seems to stimulate PRL release at a suprapituitary site and this action probably involves u opiate receptors, because naloxone prevents these stimulatory effects. The inhibitory effect of FK 33-824 on ACTH release, however, is mediated via a direct effect at the pituitary level, which does not involve u receptors, as naloxone did not prevent this effect. In this respect, its action differs from that of β-endorphin, which does not directly affect ACTH release by the anterior pituitary gland.     

Growth hormone and prolactin secretion in hypophysial stalk-transected pigs as affected by growth hormone and prolactin-releasing and inhibiting factors.

Control of growth hormone (GH) and prolactin (PRL) release was investigated in hypophysial stalk-transected (HST) and stalk-intact pigs by determining the effects of analogs of GH-releasing factors (GHRF), somatostatin (SRIF), arginine, thyrotropin-releasing hormone, alpha-methyl-rho-tyrosine, and haloperidol. HST and control gilts were challenged with intravenous injections of human pancreatic GHRF(1-40)OH, thyrotropin-releasing hormone, and analogs of rat hypothalamic GHRF. HST animals remained acutely responsive to GHRF by releasing 2-fold greater quantities of GH than seen in controls. This occurred in spite of a 38% reduction in pituitary gland weight and a 32 and 55% decrease in GH concentration and total content. During SRIF infusion, GH remained at similar basal concentrations in HST and control gilts, but increased immediately after stopping SRIF infusion only in the controls. Releasable pituitary GH appears to accumulate during SRIF infusion. GHRF given during SRIF infusion caused a 2-fold greater release of GH than seen in animals receiving only GHRF. Arginine increased (P less than 0.05) GH release in controls, but not in HST gilts, which suggests that it acts through the central nervous system. Basal PRL concentrations were greater (P less than 0.05) in HST gilts than in control gilts. TRH acutely elevated circulating PRL (P less than 0.001) in HST gilts, suggesting that it acts directly on the pituitary gland. Haloperidol, a dopamine receptor antagonist, increased circulating PRL in controls but not in HST animals. alpha-Methyl-rho-tyrosine did not consistently increase circulating PRL, however, suggesting that it did not sufficiently alter turnover rate of the tyrosine hydroxylase pool. The results indicate that the isolated pituitary after HST remains acutely responsive to hypothalamic releasing and inhibiting factors for both GH and PRL release in the pig.     

Possible direct effect of serotonin on pituitary prolactin secretion: in vivo and in vitro studies

In this work we analyze the possibility of serotonin (5-HT)-releasing prolactin (PRL) through a direct action at the pituitary level. 5-HT (2 mg/kg i.v.) stimulates PRL secretion in hypophysectomized autotransplanted animals (HAG) significantly and this effect was not influenced by pretreatment with the dopaminergic antagonist domperidone. In perifused pituitaries, 5-HT administration (0.01, 0.1 and 1 microM for 90 min, or 1, 10, 100 microM for 15 min) was ineffective in stimulating PRL release. In pituitaries obtained from animals previously treated with the neurotoxic 5,7-dihydroxytryptamine (5,7-DHT) or vehicle and incubated in the presence of 5-HT (2.5, 5 and 10 microM), no response in PRL secretion was observed. These results suggested that 5-HT does not release PRL through a direct pituitary action, and that the effect observed in HAG animals could be mediated through the release of a PRL-releasing factor after 5-HT administration.     

Effects of in vivo pretreatment with D-Trp-6-LH-RH on prolactin and LH secretion by pituitary glands in vitro

In order to study a possible direct action of LH-RH analogs on the pituitary lactotrophs, we investigated the effect of long-term in vivo pretreatment with D-Trp-6-LH-RH on in vitro secretion of PRL and luteinizing hormone (LH) by the pituitary glands from male and female rats. In vivo pretreatment with D-Trp-6-LH-RH (50 micrograms/day, SC) for 15 days greatly reduced basal in vitro PRL release (p less than 0.01) in female, but not in male pituitary glands. TRH-stimulated PRL secretion was not affected by pretreatment with D-Trp-6-LH-RH in female rats, but was impaired in male pituitaries. Acute in vitro exposure to D-Trp-6-LH-RH did not modify PRL secretion by female pituitary glands pretreated in vivo with the analog. However, this same in vivo pretreatment greatly decreased PRL release from male pituitaries (p less than 0.01). Basal in vitro LH release by male pituitary glands was partially lowered by in vivo pretreatment with D-Trp-6-LH-RH, as compared to controls (p less than 0.01), while basal LH release in female pituitaries remained at control levels. Finally, D-Trp-6-LH-RH-induced stimulation of in vitro LH release was severely impaired in female pituitaries (p less than 0.01) but only slightly reduced in the males.     

Effects of melatonin injections on the ability of golden hamster pituitaries to secrete prolactin and luteinizing hormone

Chronic afternoon (PM) but not morning injections of melatonin (MEL) induced significant reductions in testicular and seminal vesicle weights as well as attenuating serum prolactin (PRL) and luteinizing hormone (LH) levels. Although there were no treatment-induced effects on hemipituitary weights, PM-Mel injections led to significant reductions in in vitro PRL secretion and tended to increase the ability of dopamine to inhibit PRL release. It was also shown that LH-releasing hormone (LHRH) could inhibit in vitro PRL release from hamster pituitaries. Basal or LHRH-stimulated LH secretion from incubated pituitaries was not affected by Mel in vivo. From these results we conclude that properly timed Mel injections do not reduce pituitary's ability to secrete LH but severely attenuate PRL secretion. These findings are similar to those observed in pituitaries from hamsters housed in short-photoperiod conditions.     

Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.     

Depletion and release of prolactin from rat pituitaries and pituitary tumors in vitro

Depletion of pituitary prolactin (PRL) and PRL release into culture medium were simultaneously examined over a 3.5- to 4.0-hr incubation period from anterior pituitary fragments obtained from Fischer-344 or Wistar-Furth female rats treated with estrogen for 5 days, in pituitary tumors induced by 8 weeks of diethylstilbestrol (DES) treatment in Fischer-344 rats and in MtTW15 pituitary tumors transplanted subcutaneously in Wistar-Furth rats for 4 weeks. Our objective was to determine if the event known as transformation, which we define as a loss in the tissue PRL content without a corresponding and equivalent increase in the medium PRL content, occurs in rat pituitary tumors. Our results indicated that transformation did not occur in vitro in rat anterior pituitary tumors induced in Fischer-344 rats by DES treatment but was present in pituitaries from Fischer-344 rats treated for 5 days with estrogen, which served as controls. We also observed in vitro transformation in the anterior pituitary of Wistar-Furth rats treated with estrogen for 5 days (controls) and in the pituitaries of Wistar-Furth rats inoculated with the MtTW15 tumor for 4 weeks, but not in the MtTW15 tumor itself. Although transformation was present in both Fischer-344 and Wistar-Furth rats treated acutely with estrogen the timing of the transformation was delayed 1-2 hr in the Fischer-344 rats compared with Wistar-Furth females. We concluded that transformation does not precede release of prolactin in rat pituitary tumors and that in normal pituitaries the mechanisms of transformation are induced differently between the strains of rats examined.     

Inhibition of prolactin and growth hormone secretion by nickel

Nickel (Ni++) is a potent inhibitor of prolactin (PRL) secretion from isolated rat pituitary quarters in vitro, suppressing both basal PRL release and the stimulation of PRL secretion due to theophylline and dibutyryl cyclic AMP. Stimulation of growth hormone (GH) secretion by synthetic GHRH is also blunted by Ni++, although basal GH release and stimulated GH release due to theophylline or dibutyryl cyclic AMP are not suppressed. Ni++ antagonizes the stimulation of both PRL and GH secretion by barium (Ba++) ion, suggesting that the inhibitory effects of Ni++ on hormone release are due to an antagonism of calcium uptake or redistribution.     

Somatostatin depletion by cysteamine: mechanism and implication for duodenal ulceration

Cysteamine (CSH) and its close derivatives deplete immunoreactive somatostatin (SS) in rat organs. The effect of CSH is dose and time dependent and reversible. Structural requirements of the analogs are the presence of either -SH or -NH2 on a two- or three-carbon alkyl molecule; both radicals together increase, whereas insertion of carboxyl abolishes potency. The duodenal ulcerogenic potency of CSH derivatives is correlated significantly with their SS-depleting activity in the gastric mucosa. The mechanism of this action of CSH is poorly understood, but it is not caused by increased release, enhanced degradation of the peptide, or selective necrosis of SS cells. It is likely that in the intracellular environment CSH causes a conformational change in the peptide that affects the antigenic and functional properties of SS.     

The effect of bombesin on basal, alpha-methyl-p-tyrosine, haloperidol, morphine, bremazocine and stress-induced prolactin secretion

Intravenously administered bombesin lowered basal PRL levels in conscious male rats and prevented the morphine, bremazocine and stress-induced PRL secretion. The same dose of bombesin had no effect on PRL levels in alpha-methyl-p-tyrosine pretreated rats and did not affect haloperidol-stimulated PRL release. These results show that bombesin given intravenously acts as an inhibitor of PRL secretion and suggests that it does not act on the lactotrope itself but rather by an increase of the inhibitory dopaminergic tone.     

Opioid modulation of thyrotropin releasing hormone induced prolactin secretion

It is known that opioids stimulate prolactin (PRL) secretion by an action on hypothalamic neurons, but in vitro studies have suggested a direct action on the lactotrophs. The present study was performed on male rats known to have little or no PRL response to TRH. A beta-endorphin (beta EP) injection in the third ventricle stimulated PRL secretion and induced furthermore a PRL secretory reaction to TRH injected intravenously 20 min later. Pretreatment with naloxone 10 min before beta EP injection abolished not only the PRL response to beta EP but also the conjugated effect of beta EP and TRH. Pretreatment with naloxone methyl bromide (Br-naloxone), a quaternary naloxone derivative, which does not cross the blood-brain barrier, had no effect on the PRL response to beta EP but prevented the conjugated effect of beta EP and TRH on PRL secretion. Pretreatment of the animals with -methyl-parathyrosine resulting in a dopamine depletion or with haloperidol, a dopamine antagonist, could not induce lactotroph responsiveness to TRH. These results suggest that beta EP in male rat sensitizes the PRL cell to TRH by a direct effect and not through an inhibition of the dopaminergic tone.     

Signal transduction mechanisms mediating rapid, nongenomic effects of cortisol on prolactin release

While the mechanisms governing genomically mediated glucocorticoid actions are becoming increasingly understood, relatively little is known with regard to the cell signaling pathways that transduce rapid glucocorticoid actions. Studies of the cultured tilapia rostral pars distalis (RPD), a naturally segregated region of the fish pituitary gland that contains a 95-99% pure population of prolactin (PRL) cells and is easily dissected and maintained in a completely defined, serum-free media, indicate that physiological concentrations of cortisol rapidly inhibit PRL release. The attenuative action of cortisol on PRL release occurs within 10-20 min, is insensitive to the protein synthesis inhibitor, cycloheximide, and mimicked by its membrane impermeable analog, cortisol-21 hemisuccinate-conjugated bovine serum albumin (BSA). Cortisol and somatostatin, a peptide known to work through membrane receptors to inhibit PRL release, rapidly and reversibly reduces intracellular free Ca(2+) (Ca(i)(2+)), and inhibits 45Ca(2+) influx and BAYK-8644 induced PRL release. Preliminary investigations show cortisol, but not somatostatin, suppresses phospholipase C (PLC) activity in PRL cell membrane preparations. In addition, cortisol and somatostatin reduce intracellular cAMP and membrane adenylyl cyclase activity. These findings indicate that the acute inhibitory effects of cortisol on PRL release occur through a nongenomic mechanism involving interactions with the plasma membrane and inhibition of both the Ca(2+) and cAMP signal transduction pathways. Cortisol may reduce Ca(i)(2+) by inhibiting influx through L-type voltage-gated channels and possibly release through a PLC/inositol triphosphate sensitive intracellular Ca(2+) pool. In addition, it is also likely the steroid inhibits adenylyl cyclase activity in events leading to reduced cAMP production and the subsequent release of PRL.     

Stimulation of prolactin release by prolactin-releasing peptide in rats.

We have previously reported a hypothalamic peptide that shows specific prolactin (PRL)-releasing activity in vitro, named prolactin-releasing peptide (PrRP). However, its activity in vivo has not yet been shown. In this study, we examined whether PrRP could induce specific PRL release in vivo using normal cycling female and male rats. Intravenous injection of PrRP31 increased plasma PRL levels in rats in a dose-dependent manner. PrRP31 (50 nmol/kg i.v.) significantly (P < 0.05) stimulated plasma PRL levels within 25 min after injection in rats in proestrus, estrus, and metestrus. A higher dose of PrRP31 (500 nmol/kg i.v.) was necessary for a significant increase in plasma PRL levels in male rats. These results clearly indicate that female rats, especially at proestrus, are more sensitive to PrRP-induced PRL secretion than male rats. The effect of PrRP on PRL release is affected considerably by the estrous cycle and sex, which suggests that PrRP sensitivity is controlled by the endogenous hormonal milieu, such as estrogen levels. PrRP31 did not affect other pituitary hormone secretions. The results indicate that PrRP shows specific PRL-releasing activity in vivo as well as in vitro and suggest that it plays an important role in the regulation of PRL release under certain physiological conditions.