Central inhibitory action of TRH on prolactin secretion in the rat

Intravenous (iv) injection of FK33-824 [( D-Ala2, MePhe4, Met-(O)5-ol]-enkephalin, 8 and 16 nmole/100 g body wt), a potent Met5-enkephalin analog, and domperidone (1.2, 2.4, and 24 nmole/100 g body wt), a dopamine antagonist, resulted in a dose-related increase in plasma prolactin (PRL) levels in urethane-anesthetized male rats. PRL release induced by FK33-824 (16 nmole/100 g body wt, iv) was inhibited by intraventricular (icv) injection of TRH (0.6 nmole/rat). DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate, 0.6 nmole/rat, icv), a TRH analog, also blunted PRL release induced by FK33-824. PRL release induced by a smaller dose of domperidone (1.2 nmole/100 g body wt, iv) was blunted by TRH and DN-1417, whereas both peptides failed to suppress elevated PRL levels induced by larger doses of domperidone. These results suggest that TRH not only stimulates PRL secretion by acting directly at the pituitary, but has an inhibitory action on PRL release through activation of the central dopaminergic mechanism.     

Inhibition of rat prostate tumor growth by an octapeptide analog of somatostatin

Analogs of a potent octapeptide analog of somatostatin (SRIF)

were synthesized. Aromatic substitutions for Tyr resulted in little change in inhibitory potency on growth hormone (GH) secretion in the rat. Substitutions for Val or (D)Trp resulted in analogs with diminished activity. Substitution of (D)Nal for (d)Phe increased duration of GH inhibition. Final weights of subcutaneously implanted prostate tumors (R3327) were 41% lower in rats treated with an N-terminal 4-chloro-(D)phenylalanyl analog as compared to vehicle treated controls. The analog had no effect on testicular weight or final plasma testosterone levels. SRIF analogs may represent an alternative treatment for prostate cancer that would be free of the untoward reproductive effects of other treatments (e.g. LH-RH or castration).     

Helodermin, but not cholecystokinin, somatostatin, or thyrotropin releasing hormone, acutely increases thyroid blood flow in the rat

In the present study, we investigated whether peptides located within the thyroid gland, but not directly found in nerve fibers associated with blood vessels, might influence thyroid blood flow. Specifically, we evaluated the effects of helodermin, cholecystokinin (CCK), somatostatin (SRIF) and thyrotropin releasing hormone (TRH) given systemically on thyroid blood flow and circulating thyroid hormone levels. Blood flows in the thyroid and six other organs were measured in male rats using 141Ce-labeled microspheres. Circulating thyrotropin (TSH) and thyroid hormone levels were monitored by RIA. Helodermin (10(-10) mol/100 g BW, i.v. over 4 min) markedly elevated thyroid blood flow (52 +/- 6 vs. 10 +/- 2 ml/min.g in vehicle-infused rats; n = 5). Blood flows to the salivary gland, pancreas, lacrimal gland and stomach (but not adrenal and kidney) were also increased during helodermin infusions. CCK, SRIF, and TRH were without effect on blood flows to the thyroid and other organs even though these peptides were tested at higher molar doses than helodermin. Helodermin, CCK, or SRIF did not affect thyroid hormone or plasma calcium levels. As expected however, plasma TSH and T3 levels were increased at 20 min and 2 h, respectively, following TRH infusions. Since helodermin shares sequence homology with VIP, we next compared the relative effects of these two peptides on thyroid and other organ blood flows. VIP (10(-11) mol/100 g BW, i.v.) was more potent in increasing blood flows to the thyroid, salivary gland, and pancreas than an equimolar dose of helodermin. This study shows that while helodermin, like VIP, has the ability to increase thyroid and other organ blood flows, it appears to be a less potent vasodilator.     

Mutagenic activity of 2-amino-N6-hydroxyadenine in the mouse spot test.

The base analogue 2-amino-N6-hydroxyadenine (AHA) was mutagenic in the spot test in (T x HT)F1 mouse embryos. Females were injected with single doses of 20 or 40 mg AHA per kg body weight on the 9th day of pregnancy. To rank the mutagenic potency of different compounds, the frequencies of genetically relevant spots induced by 1 mg/kg body weight were calculated. The observed somatic mutation frequency for 1 mg/kg AHA was lower (1.95 x 10(-3)) spots of genetic relevance) than that of mitomycin C (16 x 10(-3)), ethylnitrosourea (6.8 x 10(-3)) and cyclophosphamide (6.4 x 10(-3)) and therefore AHA was not classified as a very potent mutagen in this test system. The doubling dose to induce genetically relevant spots was calculated to be 20 mg/kg b.w. Based on these data, AHA is suggested to be a candidate to induce recessive specific-locus mutations in germ cells of mice.     

Potent central nervous system action of p-Glu-His-(3,3'-dimethyl)-Pro NH2, a stabilized analog of TRH, to stimulate gastric secretion in rats

Intracisternal injection of the TRH analog RX 77368 (p-Glu-His-(3,3'-dimethyl)-Pro NH2) increased gastric acid and pepsin output in conscious pylorus-ligated rats. In urethane-anesthetized, gastric fistula rats, intracisternal RX 77368 or TRH induced stimulation of gastric acid output which was rapid in onset, long lasting, and dose-dependent, in doses ranging from 3 to 100 ng/rat for RX 77368, and 0.1 to 1 micrograms/rat for TRH. Vagotomy or atropine pretreatment reversed RX 77368 gastric secretory response. The analog was less effective when infused intravenously (1-10 micrograms X kg-1 X h-1) and 22 times more potent than TRH when given intracisternally. These results demonstrated the ability of RX 77368 to act within the rat brain to enhance gastric secretion (acid and pepsin) through vagus cholinergic dependent mechanisms. The enhanced potency and extended duration of action of RX 77368 over TRH, could make intracisternal injection of this peptide a useful test to induce centrally mediated vagal dependent stimulation of gastric secretion in rats.     

Acute and chronic cardiovascular effects of intermittent hypoxia in C57BL/6J mice.

We investigated the effects of 1) acute hypoxia and 2) 5 wk of chronic intermittent hypoxia (IH) on the systemic and pulmonary circulations of C57BL/6J mice. Mice were chronically instrumented with either femoral artery or right ventricular catheters. In response to acute hypoxia (4 min of 10% O2; n = 6), systemic arterial blood pressure fell (P < 0.005) from 107.7 +/- 2.5 to 84.7 +/- 6.5 mmHg, whereas right ventricular pressure increased (P < 0.005) from 11.7 +/- 0.8 to 14.9 +/- 1.3 mmHg. Another cohort of mice was then exposed to IH for 5 wk (O2 nadir = 5%, 60-s cycles, 12 h/day) and then implanted with catheters. In response to 5 wk of chronic IH, mice (n = 8) increased systemic blood pressure by 7.5 mmHg, left ventricle + septum weight by 32.2 +/- 7.5 x 10(-2) g/100 g body wt (P < 0.015), and right ventricle weight by 19.3 +/- 3.2 x 10(-2) g/100 g body wt (P < 0.001), resulting in a 14% increase in the right ventricle/left ventricle + septum weight (P < 0.005). We conclude that in C57BL/6J mice 1) acute hypoxia causes opposite effects on the pulmonary and systemic circulations, leading to preferential loading of the right heart; and 2) chronic IH in mice results in mild to moderate systemic and pulmonary hypertension, with resultant left- and right-sided ventricular hypertrophy.     

Inhibitory effect of nociceptin on somatostatin secretion of the isolated perfused rat stomach

The heptadecapeptide nociceptin/orphanin FQ (N/OFQ) has recently been isolated from porcine and rat brain and identified as the endogenous ligand of the N/OFQ receptor (NOP). It shows structural similarity with opioid peptides. N/OFQ has also been demonstrated in the gastrointestinal tract, where it inhibits gastrointestinal motility. The effect of N/OFQ on gastric neuroendocrine function is unknown as yet.In the isolated perfused rat stomach, N/OFQ 10(-6) M shows a small, but not significant decrease of basal somatostatin (SRIF) secretion. At the doses of 10(-12) M, 10(-10) and 10(-8) M N/OFQ has neither an effect on basal SRIF nor on basal vasoactive intestinal polypeptide (VIP), gastrin, substance P or bombesin secretion, respectively. However, gastric inhibitory polypeptide (GIP) 10(-9) M prestimulated SRIF secretion is significantly inhibited by N/OFQ 10(-8) M (-45+/-11%; p<0.05 vs. GIP). During concomitant infusion of the specific competitive NOP receptor antagonist [Nphe(1)]nociceptin(1-13)NH(2) 10(-6) M, the effect of N/OFQ is abolished (6+/-11%; p<0.05 vs. GIP and N/OFQ) while the opiate receptor antagonist naloxone 10(-6) M has no significant effect (-32+/-9%; ns vs. GIP and N/OFQ). At the higher concentration of N/OFQ 10(-6) M, the inhibition of prestimulated SRIF secretion (-58+/-6%; p<0.05 vs. GIP) is not influenced by the NOP receptor antagonist at the concentration of 10(-6) M (-49+/-9%; ns vs. GIP and N/OFQ) and 10(-5) M (-69+/-10%; ns vs. GIP and N/OFQ), respectively. On the other hand, infusion of naloxone 10(-6) M attenuates the inhibitory effect of N/OFQ 10(-6) M significantly (-21+/-6%; p<0.05 vs. GIP and N/OFQ).Thus, N/OFQ is an inhibitor of gastric somatostatin secretion. At the lower dose, this effect is transmitted via NOP receptors, while at the higher dose of 10(-6) M, the effect is at least in part mediated via opiate receptors.     

Urethane-induced somatostatin mediated inhibition of gastric acid: reversal by the somatostatin 2 receptor antagonist, PRL-2903.

Urethane increases the release of somatostatin (SRIF) which inhibits gastric acid secretion. The SRIF monoclonal antibody, CURE.S6 and the novel sst2 antagonist, PRL-2903 injected intravenously at maximal effective doses increased gastric acid secretion by 2 and 10 fold respectively from basal values within 30 min in urethane-anesthetized rats. Plasma gastrin levels were elevated 2.5 fold within 15 min by PRL-2903 (1.3 micromol/kg, i.v.). These data indicate that the low gastrin and acid secretion levels induced by urethane result from endogenous SRIF acting on sst2 and that PRL-2903 is a valuable SRIF antagonist to assess sst2 mediated events.     

Octapeptide analogs of somatostatin exhibiting greatly enhanced in vivo and in vitro inhibition of growth hormone secretion in the rat

Analogs of a superactive somatostatin (SRIF) octapeptide (code named SMS 201-995 (1)) were synthesized using solid-phase synthetic methodology and assayed for their ability to inhibit growth hormone release from cultured rat anterior pituitary cells and in sodium pentobarbital-anesthetized rats. One analog: (Formula: see text) exhibited greatly enhanced in vitro inhibitory activity (greater than 1,000x) relative to both the parent octapeptide molecule and to the 14 amino acid SRIF molecule. This analog which was also very potent in vivo contains a tyrosine residue and, given its high in vitro activity, may be of investigative importance as a radioiodinated ligand in receptor assays. An octapeptide retro-inverso analog also exhibited significant SRIF-like activity. Several very low activity octapeptide analogs were synthesized and were found to be devoid of SRIF-antagonist activity. A dodecapeptide analog previously shown to be superactive in vivo also demonstrated high in vitro activity.     

Influence of hydrocortisone acetate administered to the lactating rat on protein and lactose content in milk and serum protein, glucose and insulin levels in dams and pups

The influence of i.m. administration to the mother of hydrocortisone acetate (doses of 0.4, 0.8 or 2.0 mg/100 g body weight/day) during the first 15 days of lactation on milk protein and lactose composition and serum levels of protein, glucose and insulin in dams and pups is studied. Total serum proteins and albumin/globulin ratio in dams were unchanged by treatment. The daily injection of 0.4 or 0.8 mg/100 g body weight failed to alter serum levels of glucose or insulin in dams, whereas a dose of 2.0 mg/100 g body weight led to a rise in glucemia (from 118 +/- 3.2 to 133 +/- 5.3) which was accompanied by a sharp change in insulinemia (from 40.7 +/- 4.1 to 83.6 +/- 6.9). All three doses raised protein levels in milk. The smallest increase was recorded with 2.0 mg/100 g body weight; this dose also reduced milk lactose content. Total serum proteins in pups rose slightly but nonsignificantly, and no significant effects were noted on albumin/globulin ratio or serum glucose and insulin levels.     

Effect of pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) on tissue oxygen content--treatment in central nervous system of mice

It has been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in preventing neuronal cell death and is also a potent vasodilator. Cerebral hypotension and hypoperfusion during cerebral ischemia and neurodegenerative diseases are well known as some of the negative factors which aggravate neuronal cell death. Nevertheless, the effect of PACAP on the cerebral circulation was not understood well. Therefore, in the present study, we determined the mean arterial blood pressure (MBP), regional cerebral blood flow (rCBF) and cerebral oxygen content (pO2) in mice, and estimated the therapeutically useful doses of PACAP. Under barbiturate anesthesia, polyethylene tubes were inserted into mice to monitor MBP and to administer PACAP (5 x 10(-13)-5 x 10(-8) mol/kg) or vasoactive intestinal peptide (VIP; 5 x 10(-12) and 5 x 10(-9) mol/kg). Then, MBP, rCBF and cerebral pO2 were simultaneously measured in the mice. PACAP (5 x 10(-10)-5 x 10(-9) mol/kg) injections transiently decreased MBP, and cerebral pO2. PACAP (5 x 10(-8) mol/kg) injections produced a long-lasting potent decline of MBP, rCBF and cerebral pO2. Therefore, PACAP should be applied at low doses which do not influence the MBP and cerebral circulation to determine the therapeutically useful doses of PACAP for neuroprotection.     

Evidence of UCP1-independent regulation of norepinephrine-induced thermogenesis in brown fat

To study the thermal response of interscapular brown fat (IBF) to norepinephrine (NE), urethan-anesthetized rats (1.2 g/kg ip) maintained at 28-30 degrees C received a constant venous infusion of NE (0-2 x 10(4) pmol/min) over a period of 60 min. IBF temperatures (T(IBF)) were recorded with a small thermistor fixed under the IBF pad. Data were plotted against time and expressed as maximal variation (Deltat degrees C). Saline-injected rats showed a decrease in T(IBF) of approximately 0.6 degrees C. NE infusion increased T(IBF) by a maximum of approximately 3.0 degrees C at a dose of 10(4) pmol x min(-1) x 100 g body wt(-1). Surgically thyroidectomized (Tx) rats kept on 0.05% methimazole showed a flat response to NE. Treatment with thyroxine (T(4), 0.8 microg x 100 g(-1) x day(-1)) for 2-15 days normalized mitochondrial UCP1 (Western blotting) and IBF thermal response to NE, whereas iopanoic acid (5 mg x 100 g body wt(-1) x day(-1)) blocked the effects of T(4). Treatment with 3,5, 3'-triiodothyronine (T(3), 0.6 microg x 100 g body wt(-1) x day(-1)) for up to 15 days did not normalize UCP1 levels. However, these animals showed a normal IBF thermal response to NE. Cold exposure for 5 days or feeding a cafeteria diet for 20 days increased UCP1 levels by approximately 3.5-fold. Nevertheless, the IBF thermal response was only greater than that of controls when maximal doses of NE (2 x 10(4) pmol/min and higher) were used. Conclusions: 1) hypothyroidism is associated with a blunted IBF thermal response to NE; 2) two- to fourfold changes in mitochondrial UCP1 concentration are not necessarily translated into heat production during NE infusion.     

The effect of atrial natriuretic peptide on small intestinal contractility and transit.

Isometric tension in response to ANF (10(-10) to 10(-4) M) was recorded from longitudinally and circularly oriented rat jejunal smooth muscle strips. Conscious, fasted rats received an IV infusion of 1.25 nmol ANF/100 g body weight in 0.5 ml normal saline and controls received saline alone. Five minutes later 10 muCi Na2 51CrO4 in 0.5 ml saline was instilled through a jejunostomy. Fifteen minutes later animals were sacrificed, and the gut divided into 8 equal segments of small intestine, cecum and remaining colon. The radioactivity of each segment was measured and a geometric center of transit determined for each group. ANF induced relaxation of longitudinally oriented strips (Tm = -72.3 +/- 10.7 mN/g, ED50 7.3 +/- 3.6 x 10(-8) M), and contraction of circularly oriented strips (Tm = 35.0 +/- 5.0 mN/g, ED50 1.3 +/- 1.0 x 10(-8) M). This response was unaffected by 10(-6) M tetrodotoxin. The geometric mean center of transit was significantly (p less than 0.001) further aboral in ANF-treated compared to control animals (intestinal segment 4.2 +/- 0.2 vs. 3.2 +/- 0.2).     

Effects of homologous ghrelins on the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus

Ghrelin is a gut-brain peptide synthesized mainly in the oxyntic mucosal cells of the stomach, and has potent growth hormone (GH)-releasing and orexigenic activities. Recently, two forms of ghrelin, ghrelin-C8 and -C10, were identified in the Mozambique tilapia (Oreochromis mossambicus). The present study describes in vitro and in vivo effects of these endogenous ghrelins on the GH/insulin-like growth factor-I (IGF-I) axis. Ghrelin-C8 (100 nM) stimulated GH release from primary cultures of pituitary cells after 4 and 8 h of incubation, whereas no effect was seen on prolactin (PRL) release. Stimulatory effects of ghrelin-C8 and -C10 (100 nM) on GH release during 6 h of incubation were blocked by pre-incubation with GHS receptor antagonist, [D-Lys(3)]-GHRP-6 (10 microM). Intraperitoneal injection of ghrelin-C8 (1 ng/g body weight) and -C10 (0.1 and 1 ng/g body weight) significantly increased plasma GH levels after 5 h. Significant increases were observed also in hepatic expression of IGF-I and GH receptor (GHR) mRNA following injections of both forms of ghrelin (0.1 and 1 ng/g body weight), although there was no effect on plasma levels of IGF-I. In the next experiment, both forms of ghrelin (1 ng/g body weight) significantly increased plasma IGF-I levels 10 h after the injection. No significant effect of either ghrelin was observed on plasma PRL levels. Both forms of GHS receptor (GHSR-1a and -1b) were found in the pituitary, clearly indicating that tilapia ghrelins stimulate primarily GH release through the GHS receptor. Stimulation of hepatic expression of IGF-I and GHR suggests metabolic roles of ghrelin in tilapia.     

Modulation of gastric acid secretion by adenosine in conscious rats

Basal (nonstimulated) gastric acid output was determined in conscious rats fitted with indwelling gastric cannulae. The adenosine deaminase resistant analog of adenosine, R-phenylisopropyladenosine, elevated intraluminal pH beyond 7.0 and decreased gastric acid secretion when given at doses of 0.10 or 1.0 mg/kg, while S-phenylisopropyladenosine at similar doses did not affect either gastric acid output or pH. The potent adenosine receptor antagonist, 8-phenyltheophylline, given at doses of 0.1, 1.0, and 2.5 mg/kg augmented gastric acid output and, at doses of 0.01, 0.1, 1.0, and 2.5 mg/kg, blocked the acid-reducing effect of R-phenylisopropyladenosine (0.1 mg/kg). These data suggest that adenosine systems may be important regulators of gastric function.     

Influence of centrally administered somatostatin and two related peptides on intestinal absorption of water and electrolytes in conscious dogs

The effects of intracerebroventricular (ICV) administration of somatostatin (SRIF) and two related peptides, anti SRIF and SMS 201-995, on jejunal fluxes of water, Na+ and K+ were investigated in dogs prepared with a Thiry-Vella (TV) loop. Intestinal transport in the TV loop and concomitant transit time were also measured during infusion (2 mg/min) of an isotonic electrolyte solution and phenol-red bolus injections. Basal net water absorption was reduced significantly (p less than 0.01) over periods of 2 to 5 hr and in a dose-related manner, with ICV administrations of SRIF (5 to 100 ng/kg); doses of SRIF, 5 to 25 times higher but administered IV, were inactive. Similar reductions in the net fluxes of water, Na+ and K+ were observed over 2 to 5 hr following ICV administration of a putative somatostatin antagonist and SMS 201-995 at doses of 100 ng/kg. Neither metoclopramide (1 mg/kg), phentolamine (0.1 mg/kg) nor methysergide (0.2 mg/kg) given IV were able to antagonize the effects of centrally administered SRIF (100 ng/kg) on intestinal fluxes. In contrast, the effects of SRIF were abolished completely by naloxone (0.2 mg/kg) but not methyl-naloxone (0.3 mg/kg) given systemically. It is concluded that somatostatin and the two related peptides act centrally to reduce jejunal absorption of water and electrolytes. The effects of SRIF appear to be related to opiate receptors, possible involving central nerve pathways which utilize opiate-like transmitters.     

The cytoprotective effect of the desamino-D-Trp 7-10 fragment of somatostatin on ethanol-induced erosions in the rat stomach

The concept of cytoprotection was established by Robert. It means that several materials of different structure and in doses which are not antisecretoric ones, can protect the gastric mucosa against exogenous noxous agents. Absolute ethanol causes haemorrhagic erosions on the rat gastric mucosa. Somatostatin (SRIF) and its 7-10 fragment, which is the active centre of the hormone were cytoprotective in the ethanol-induced cytoprotective model. The desamino-D-Trp fragment (a suspected enzyme resistant variant of the active centre of SRIF) is also protective. It decreases significantly the extension of the lesions. Its dose-response curve shows two peaks, which may correspond to the existence of two SRIF receptor types, described in the rat gastric mucosa.     

Comparison of the effect of various doses of thyroxine on jejunal disaccharidases in intact and adrenalectomized rats during the first 3 weeks of life.

Administration of D,L-thyroxine (T4) daily (25 or 250 nmol/100 g body weight/24 h) for 4 days increased jejunal activity in 15-day-old rats; no effect was seen in 8-day-old rats. Maltase activity was increased in 15-day-old rats only when a 250-nmol dose was used. Similar results were obtained in unoperated and adrenalectomized rats. Specific activity of lactase was not influenced in unoperated 8- and 15-day-old rats; it was decreased in adrenalectomized 8- and 15-day-old rats when treated with a 250-nmol dose of T4; in 15-day-old rats, it also responded to 25-nmol doses. The lowest dose used (2.5 nmol/100 g body weight) had no effect at all.     

Responses of giant freshwater prawn (Macrobrachium rosenbergii) to challenge by two strains of Aeromonas spp

The virulence of two Aeromonas strains (A. veronii and A. caviae) isolated from the hepatopancreas of apparently healthy giant freshwater prawns (Macrobrachium rosenbergii) was compared using a challenge by injections. For the A. veronii strain, challenge with 3.7 x 10(5) cells/g of body weight led to 100% mortality; for the A. caviae strain, 3.8 x 10(6) cells/g produced 100% mortality. The 50% lethal doses (LD50) were 2.0 x 10(3) cells/g for A. veronii and 51.2 x 10(3) cells/g for A. caviae. Use of different culture media (trypticase soy broth vs prawn muscle extract) did not significantly affect the virulence of A. veronii. Injection of a sublethal dose (1 x 10(3) cells/g) of A. veronii led to a significant decrease in the total hemocyte count (THC) between 4 and 24 h after injection. Saline injections also caused a similar though less decrease in THC. In the first 24 h after injection of A. veronii (1 x 10(3) cells/g), the change in the percentages of granulocytes (both granular cells and semigranular cells) in the hemolymph was significantly different. After a significant initial increase, the percentage of hyaline cells fell by a factor of 4, from 9 to 2%. Phenoloxidase activity increased fourfold immediately after injection and returned to preinjection levels at 24 h.