Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.     

Effects of prostaglandin E2 and DL204 IT,an inhibitor of prostaglandin degradation,on ovulation and ovum transport in the hamster

The effects of 2(3-ethoxyphenyl)-5,6-dihydro-s-triazole-[5,1-a] isoquinoline (L-11204 or DL 204 IT) and PGE₂ on ovulation and ova transport were studied. DI 204 IT was administered in doses of 0.2–25 mg/Kg s.c. on the day of estrus. A small reduction in ovulating follicles was observed 96 hours later, but only at the 5 mg/Kg dose level. At all dose levels, however DL 204 IT caused a dose-related reduction in the number of ova in the oviducts. PGE₂ at a total dose of 2 mg/animal s.c., administered in 4 divided doses over the second and third day of the cycle did not affect ovulation or ova transport. PGE₂ plus DL 204 IT (5 mg/Kg), however, completely blocked ovulation in all but one animal. The animal had one ovulated follicle and a single ova was recovered from its oviduct.     

Effects of luteinizing hormone, progesterone, testosterone, estradiol and corticosterone on ovulation and luteinizing hormone release in hens treated with aminoglutethimide

Aminoglutethimide (AG), an inhibitor of steroidogenesis, was administered s.c. to 5 groups of laying hens at a dose of 200 mg AG/kg body weight 9 h before expected midsequence ovulation. This dose has previously been demonstrated to consistently block ovulation. The injection of AG was followed by s.c. injections of: Group 1, 1.0 mg progesterone; Group 2, 0.1 mg estradiol-17 beta; Group 3, 1.5 mg corticosterone, all at 6 h prior to expected ovulation; Group 4, 1.0 mg testosterone at both 8 h and 5 h before expected ovulation; and Group 5, 25 micrograms of ovine luteinizing hormone (LH) at 8 and 50 micrograms ovine LH at 6 h before expected ovulation. For each group, 4 control hens were injected with AG and the appropriate vehicle. Blood samples were taken at 1- or 2-h intervals from the time of AG injection to the expected time of ovulation. The hens were killed 4 h after expected ovulation and examined for the occurrence of ovulation. In all hens injected with vehicle, ovulation and the preovulatory surges of progesterone, testosterone, estradiol-17 beta and LH were inhibited. The plasma concentration of corticosterone was not reduced following an injection of AG. Four of 6 hens ovulated in response to injection of ovine LH, although neither endogenous LH nor progesterone were released. Thus, LH appears to play a direct role in follicular rupture and extrusion of the ovum. The administration of progesterone induced a significant and prolonged rise in LH, restoring AG-blocked ovulation in all hens treated (n = 6). Injections of testosterone restored LH release in all hens and ovulation in 2 of 7 hens treated. Three of 7 hens ovulated in response to the corticosterone injection. A preovulatory rise in LH was not observed, indicating that corticosterone may exert its ovulation-inducing effect directly on the mature follicle. Estradiol-17 beta did not restore LH release or ovulation in any of the hens treated with AG.     

Induction of luteal regression in the marmoset monkey (Callithrix jacchus) by a gonadotrophin-releasing hormone antagonist and the effects on subsequent follicular development

Doses of 100 or 200 micrograms of a novel GnRH antagonist ([N-acetyl-D beta Na11-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8-D-Ala10]NH2 GnRH) (4 animals/dose) were administered on Days 10/11 of the luteal phase and induced a marked suppression of circulating bioactive LH and progesterone concentrations within 1 day of treatment (P less than 0.01). Thereafter, progesterone concentrations remained low or undetectable until after the next ovulation. Similar results were obtained when 200 micrograms antagonist were given on Days 5/6 of the luteal phase (N = 4). The interval from injection of antagonist (200 micrograms but not 100 micrograms) to ovulation (based on a rise in progesterone above 10 ng/ml) was significantly longer than that from prostaglandin-induced luteal regression to ovulation in control cycles (N = 4/treatment) (range, 13-15 days after antagonist vs 8-10 days after prostaglandin, P less than 0.01). This delay of 4-5 days was equivalent to the duration for which LH concentrations were significantly suppressed by 200 micrograms antagonist when administered to ovariectomized animals (N = 3). Corpus luteum function during the cycle after GnRH antagonist treatment appeared normal according to the pattern of circulating progesterone. These results show that corpus luteum function and preovulatory follicular development in the marmoset monkey are dependent on pituitary gonadotrophin secretion.     

Effect of various agents on ovarian plasminogen activator activity during ovulation in pregnant mare's serum gonadotropin-primed immature rats

The plasminogen activator/plasmin synthetic substrate S-2251 was used to measure the effect of indomethacin, cycloheximide, colchicine, dexamethasone, tranexamic acid, and aprotinin on the elevation of ovarian plasminogen activator (PA) that normally occurs during ovulation in the rat. Young Wistar rats were weaned on the morning of Day 21, given 4.0 IU of pregnant mare's serum gonadotropin (PMSG) s.c. at 0800 h on Day 22, and given 10.0 IU of human chorionic gonadotropin (hCG) on Day 24. These animals normally began ovulating between 0000 and 0200 h on Day 25. The induced ovulation rate was 11.5 +/- 2.2 ova/rat, based on the number of ova in the oviducts of control animals at 0900 h on Day 25. In the controls, PA activity in extracts of homogenized ovaries increased 3-fold from 0.125 +/- 0.010 OD units just before the administration of hCG to 0.371 +/- 0.021 at 12 h after hCG, i.e., near the time of ovulation. Indomethacin, in doses of 0.1-1.0 mg/rat, inhibited ovulation but did not inhibit the normal increase in PA activity, whereas indomethacin at the high dose of 10.0 mg/rat inhibited both ovulation and PA activity. Cycloheximide, at a dose of 0.1 mg/rat, was given at 12 h before hCG, immediately after hCG, and at 9 h after hCG. This agent inhibited ovulation most effectively when given at 12 h before hCG, yet it inhibited PA activity most effectively when given immediately after or at 9 h after hCG. Colchicine, at a dose of 0.1 mg/rat, inhibited ovulation, but not PA activity, when it was given 1 h before hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformationally restrained cyclic peptides as antagonists of luteinizing hormone-releasing hormone

Using minimum energy calculations and molecular dynamics techniques the preferred conformational states of LHRH and its analogues have been reported to involve a modified beta-bend between residues 5 to 8. Based on some of these models cyclic peptide analogues of LHRH antagonists were synthesised using solid phase peptide synthesis methodology. The analogues were tested for their ability to inhibit ovulation in normal cycling rats. Some analogues were also tested in receptor binding and in vitro LH release assays. The most potent cyclic peptide analogue, Ac-D-Phe(p-C1)-D-Phe(p-C1)-D-Trp-Ser-Glu-D-Arg-Leu-Lys-Pro-D-Ala-NH2 (V), had an ED50 value of 91.9 micrograms/kg in the inhibition of ovulation test. The corresponding linear peptide (IV) was about three times less potent. Analogues with smaller or larger ring sizes or with modifications within the ring were also prepared but these were either less potent or inactive, up to a dose of 1000 micrograms/kg, in inhibiting ovulation in normal cycling rats.     

Properties of a potent LHRH antagonist (Org 30850) in female and male rats.

Org 30850 (Ac-D-pClPhe1,2,D-Bal3,D-Lys6,D-Ala10-LHRH) is a novel LHRH antagonist, which is being developed for the treatment of hormone-dependent disorders. The activities of this compound with respect to its endocrinological properties and side-effects were tested in rats and the results were compared with one of the first LHRH antagonists: Ac-D-pClPhe1,2,D-Trp3,D-Arg6,D-Ala10-LHRH (Org 30276). A single subcutaneous (s.c.) dose of 0.3 micrograms/kg Org 30850 administered to rats in pro-estrus gave inhibition of ovulation in approx. 50% of the rats, whereas Org 30276 was approx. 4 times less potent. The effect of a single s.c. injection of Org 30850 on testosterone levels in young adult male rats was also studied. The administration of 250 micrograms/kg or higher of Org 30850 induced a significant decrease in testosterone levels after 3 h, this effect lasted for at least 48 h. Treatment of female rats for 14 days with a daily dose of 12 micrograms/kg Org 30850 decreased statistically significantly uterine and ovarian weights. At a daily dose of 50 micrograms/kg Org 30850 completely suppressed estrous cycles and significantly decreased estradiol and FSH serum levels. The LH levels were below the detection level in both control and treated animals on the (expected) second day of di-estrus. Treatment of male rats for 14 days (25-200 micrograms/kg) resulted in a dose-dependent reduction of the gonads, accessory sex organs, testosterone levels and gonadotrophins. The decrease in gonadal function in both sexes was reversible since the females proved to be as fertile as the controls 6 weeks after the last treatment and an almost complete recovery of the weight of testes, seminal vesicles and ventral prostate was observed in the males 4 weeks after cessation of treatment. In contrast to Org 30276, Org 30850 exerted very slight irritation at the site of injection and no edematous reactions in the extremities at a daily dose of up to 8 mg/kg in male rats. It is concluded that Org 30850 is a very potent LHRH antagonist without edematous reactions and with a more favourable therapeutic index than Org 30276.     

Epostane and indomethacin actions on ovarian kallikrein and plasminogen activator activities during ovulation in the gonadotropin-primed immature rat.

Kallikrein and plasminogen activator (PA) are serine proteases that have been implicated in the ovulatory process. Epostane and indomethacin are anti-ovulatory agents that inhibit steroid and eicosanoid synthesis, respectively. This study examines the effects of these two anti-ovulatory agents on ovarian kallikrein and PA activities during ovulation. The proteases were assayed by their actions on chromogenic peptide substrates S-2266 and S-2251, respectively. The ovulatory process was induced in 25-day-old Wistar rats by giving them hCG (10 IU, s.c.) 2 days after the animals had been primed with eCG (10 IU, s.c.). Control animals ovulated approximately 60-70 ova/rat, with the first ova appearing in the oviducts at 10-12 h after hCG administration, and this was the same time ovarian kallikrein and PA activities reached a peak. When doses of epostane ranging from 0.1-5.0 mg/rat or doses of indomethacin ranging from 0.03 to 3.16 mg/rat were administered s.c. at 3 h after hCG, the two drugs inhibited ovulation and ovarian kallikrein and PA activities in a dose-dependent manner. However, the anti-ovulatory action of the two drugs was more closely correlated with suppression of kallikrein activity than with PA activity. Treatment of the animals with exogenous progesterone reversed the inhibitory action of epostane, but not of indomethacin. The results suggest that the increase in ovarian progesterone at the time of ovulation may influence ovarian kallikrein and PA activities.     

Effect of prolonged administration of long-acting somatostatin on caerulein, CCK-8 and GRP induced pancreatic growth in the rat.

This work investigates the effects of the long-acting somatostatin analogue, octreotide also named SMS 201-995 or Sandostatin, on pancreatic growth in function of the dose and duration of treatment. Octreotide was administered s.c. twice daily, while pancreatico-trophic peptides, caerulein and CCK-8 (1.8 nmol/kg b.wt.) or GRP (3.6 nmol/kg b.wt.) were administered s.c. three times daily. Octreotide (1,10,20 micrograms/kg b.wt.) administered for 4 days reduced pancreatic growth induced by caerulein in a dose-dependent manner. This effect, significant from 10 micrograms/kg, was more obvious with 20 micrograms/kg. At this latter dose, octreotide inhibited significantly the increase in pancreatic weight and protein, RNA, DNA and enzyme content induced by a 4- or 10-day treatment with GRP. A similar effect was observed after a 4-day treatment with CCK-8, but after a 10-day treatment only protein and enzyme contents were reduced. Octreotide by itself did not affect pancreatic size and composition after a 10-day treatment, but decreased enzyme content after a 4-day treatment. It is concluded that octreotide exerts an antitrophic effect on the rat exocrine pancreas which depends on the dose and duration of treatment and can be modulated by the trophic factor applied for a long-term.     

Central cardiovascular and thermal effects of prostaglandin F2 alpha in rats.

Administration of PGF2 ALPHA (0.2--6.4 micrograms) into the lateral cerebral ventricle (i.c.v.) induced dose-dependent increases in blood pressure, heart rate and body temperature in urethane-anaesthetised rats, but had no effect on these parameters when the same dose range was administered intravenously. Peripheral pretreatment with sodium meclofenamate (50 mg/kg s.c.) shifted all the dose-response curves for PGF2 alpha (i.c.v.) to the left, but indomethacin (50 mg/kg s.c.) did not significantly affect those changes. Central pretreatment with sodium meclofenamate or indomethacin (1.25 mg per rat i.c.v.) failed to modify significantly the effects of centrally administered PGF2 alpha. The results support previous suggestions that PGF2 alpha may participate in the central control of the cardiovascular and thermoregulatory systems, and also suggest that there may be differences in the sites and/or modes of action between sodium meclofenamate and indomethacin.     

Centrally administered [D-Trp11]neurotensin, as well as neurotensin protected from inactivation by thiorphan, modifies locomotion in rats in a biphasic manner

Neurotensin injected intracerebroventricularly at the dose of 30 ng per rat was without intrinsic effect on locomotion. When associated with the enkephalinase inhibitor thiorphan (50 micrograms, intracerebroventricular) it decreased locomotor activity. On the contrary, the 3 micrograms dose of NT, which had a tendency to decrease locomotion, stimulated locomotor activity when associated with thiorphan (50 micrograms, intracerebroventricular). This effect was independent of endogenous enkephalins since it was not suppressed by a high dose of naloxone (2 mg/kg). Similarly, increasing doses of the enkephalinase-resistant peptide [D-Trp11]neurotensin had a biphasic effect on locomotion since doses lower than 60 ng were hypokinetic whereas higher doses were hyperkinetic. This latter effect was not modified by thiorphan. It was antagonized by the dopamine antagonist haloperidol (50 micrograms/kg, IP).     

Long-term facilitation of social recognition in rats by vasopressin related peptides: a structure-activity study.

The effect of several vasopressin-related peptides was investigated in the social recognition paradigm, that consists of two successive encounters of a resident and a juvenile rat. The decrease of social investigation time of the resident rat during the second encounter served as a measure for social recognition. Single administration (3.0 micrograms, s.c.) of the vasopressin (AVP)-related peptides AVP-(1-8), AVP-(1-7) or AVP-(1-6), injected just after the first encounter, resulted in social recognition after 24 hours. Such an effect was not observed after placebo treatment or an injection with AVP-(1-5), [pGlu4, Cyt6]AVP-(4-8), [pGlu4, Cyt6]-AVP-(4-9), AVP-(7-9) or oxytocin-(1-6)-NH2. The peptide AVP-(1-6) was also active when administered in a dose of 0.3 micrograms in contrast to other peptides. Thus, vasopressin related peptides induce long term facilitation of social recognition and this action resides in the covalent ring structure of vasopressin. This effect resembles the vasopressin-induced facilitation of particular memory processes, as revealed with other behavioral paradigms.     

Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist-[N-Ac-D-2-Nal1, D-4-Cl-Phe2, D-3-Pal3, Arg5, D-A26, D-Ala10]-LHRH inhibited ovulation completely at 1 micrograms/rat and three of the other antagonists showed some antiovulatory activity at 0.5 micrograms/rat.     

Blocking of cholecystokinin octapeptide behavioral effects by proglumide

An open field apparatus was used to assess the effect of proglumide, a selective antagonist of cholecystokinin octapeptide (CCK-8), to block the behavioral effect of CCK-8 in rats. Intracerebroventricular (ICV) injection of CCK-8 (0.5 to 2 micrograms) was effective in suppressing general exploratory activities and this effect was blocked by proglumide at doses of 2 to 5 micrograms administered ICV or 1 mg/kg administered subcutaneously. The effect of peripherally administered CCK-8 (10 micrograms/kg) was blocked by peripherally administered proglumide at a dose of 2 mg/kg but not by centrally administered proglumide at a dose of 5 micrograms/rat. The behavioral effect of CCK-8 was thus clearly blocked by proglumide.     

Inhibition of ovulation in PMSG/hCG-treated immature rats by rotenone, a specific inhibitor of mitochondrial oxidation

Immature Wistar rats were induced to ovulate by treatment with PMSG and hCG. Control animals ovulated 43.5 +/- 0.36 ova/rat. Intraperitoneal injection of rotenone doses of 0.125, 0.25 and 0.50 mg/kg reduced the ovulation rate to 24.0 +/- 3.08, 8.0 +/- 0.88 and 1.5 +/- 0.44 ova/rat, respectively. The rotenone significantly reduced ovarian cytochrome oxidase activity and progesterone production, but not production of oestradiol or testosterone. Thyroxine treatment at a dose of 5 mg/kg s.c. reversed the rotenone inhibition of ovulation. The results suggest that an increase in mitochondrial respiration is an essential feature of the ovulation process in mammals.     

Possible involvement of lipoxygenase products of arachidonic acid pathway in ovulation

The possible involvement of products of the lipoxygenase pathway of arachidonic acid cascade in ovulation was tested by intrabursal injection of nordihydroguaiaretic acid (NDGA); 5, 8, 11-eicosatriynoic acid (5, 8, 11-ETYA), 3 amino-1-(3 trifluromethyphenyl)-2-pyrazoline hydrochloride (BW755c) and (FPL 55712). All these drugs reduced the number of ova released from the treated ovaries in a dose-dependent manner, without affecting ovulation from contralateral ovaries. NDGA was most potent since it completely blocked ovulation from the treated ovaries in 17/38 rats receiving a dose higher than 0.15 mg/bursa. This effect of NDGA cannot be ascribed to its inhibition of ovarian PGE synthesis. Conversion of labeled arachidonic acid via the lipoxygenase pathway by preovulatory rat follicles was demonstrated by TLC chromatography. Collectively, these results suggest the involvement of products of lipoxygenase pathway of arachidonic acid in ovulation in the rat.     

Influence of oxytocin on feeding behavior in the rat

Oxytocin, whether administered intraperitoneally (IP) (375-6,000 micrograms/kg) or intracerebroventricularly (ICV) (1-10 micrograms/rat), dose-dependently reduced food consumption and time spent eating and increased the latency to the first meal in rats fasted for 21 hr. Pretreatment with the oxytocin antagonist d(CH2)5Tyr(Me)-[Orn8]vasotocin (ICV 10 micrograms/rat) completely prevented the feeding inhibitory effect of an equal dose of ICV oxytocin, and per se increased food intake. Our data further support the hypothesis that oxytocin plays the role of neurotransmitter or neuromodulator in the CNS, and suggest that its involvement in a number of homeostatic systems may include appetite control.     

Relevance of mating-associated stimuli, ovulation, and the progesterone receptor for the post-coital inhibition of estrous behavior in the female rabbit

Estrous female domestic rabbits (Oryctolagus cuniculus) display scent marking (“chinning”) and sexual receptivity. Mating induces ovulation, which occurs approximately 12 h later, and also decreases chinning and receptivity. In the present study, we explored the participation of mating-associated stimuli, ovulation, and the progesterone receptor (PR) in mediating such behavioral effects. We found that copulatory stimuli were not necessary, and that ovulation alone was sufficient, as these behavioral changes were replicated in unmated females by intravenous administration of human chorionic gonadotropin (hCG). The post-mating administration (s.c.) of 5 μg/day estradiol benzoate (EB), prevented the decline in chinning and receptivity. A lower dose of EB (1 μg/day) had no effect, nor did the antiprogestin RU486 (20 mg, s.c., administered 3 h before mating). However, the combination of a single pre-mating administration of RU486 plus the post-mating administration of 1 μg/day EB completely blocked the decline in estrous behavior. We propose that PR activation around the time of mating and a post-mating decline in ovarian estradiol secretion and/or estradiol responsiveness act in parallel to terminate estrus in this species.     

The effect of indomethacin on HMG-HCG induced ovulation in the rhesus monkey

The investigation was designed to study the influence of indomethacin on gonadotropin induced ovulation in the rhesus monkey. Six mature female monkeys were treated with HMG-HCG for at least 2 control ovulatory cycles at dosage levels adjusted to induce ovulation while avoiding superovulation. Ovulation was confirmed by observation of the ovaries for fresh ovulation points at laparotomy. Following establishment of an appropriate dosage schedule, treatment was begun with indomethacin (5 mg/kg/day) starting 5 days prior to HCG and continuing to the time of laparotomy. In a second treatment cycle, indomethacin was administered at a dose of 5 mg/kg b.i.d. together with the established dose of HMG-HCG. Ovarian inspection was carried out as in the control cycles. Venous blood was obtained on treatment days 4, 7, 10 and 11 for determination of serum estrone, estradiol and progesterone. Indomethacin administration resulted in ovulation inhibition at a dose of 10 mg/kg/day when ovulation inducing doses of gonadotropins were administered. Peripheral blood steroid levels suggest that follicle maturation and estrogen production are unimpaired by indomethacin. These findings indicate that the ovarian synthesis of prostaglandin may be essential in the process of ovulation.     

CCK-antagonist L-364,718: influence on rat pancreatic growth induced by caerulein and bombesin-like peptides

The present study investigates the inhibitory effect of the novel potent benzodiazepine-related CCK-antagonist L-364,718 on pancreatic growth in the rat induced by chronic administration of caerulein and bombesin-like peptides. Caerulein, injected s.c. twice daily at a dose of 1 microgram/kg body weight, and bombesin (10 micrograms/kg) induced a similar increase (1.5-3-fold) in pancreatic wet weight, total protein, amylase, trypsin, putrescine and spermidine content after 14 days of treatment. Growth induced by caerulein showed a significant increase in total DNA content suggesting cellular hyperplasia, whereas bombesin-like peptides led to cellular hypertrophy. In comparison to bombesin the decapeptide neuromedin C (10 micrograms/kg) was found to be 30-50% less potent. In the same dose range, neuromedin B and the tachykinins neurokinin A and B, all structurally related to bombesin, had no significant trophic effect on the rat pancreas. Administration of the CCK-antagonist L-364,718 twice daily at a dose of 0.1 mg/kg or at 1.0 mg/kg, either s.c. or orally, led dose-dependently to a near-complete inhibition of the caerulein-induced trophic effect. In contrast, L-364,718 administered at identical dosages, did not affect pancreatic hypertrophy induced by bombesin and neuromedin C. It is concluded that both peptides mediate their effect on the rat pancreas directly and not via release of endogenous cholecystokinin. Tachykinins are not involved in the regulation of pancreatic growth. Caerulein- and bombesin-like peptides have comparable effects on the stimulation of protein and polyamine synthesis.