Differential effect of fluorinated analogs of TRH on the cardiovascular system and prolactin release

The effects of thyrotropin-releasing hormone (TRH) and the TRH-analogs, 4-fluoro-Im-TRH (4-F-TRH) and 2-trifluoromethyl-Im-TRH (2-TFM-TRH), on the cardiovascular system and prolactin (PRL) release were examined in conscious rats. TRH (2.8 or 28 nmol) injected into the anterior hypothalamus produced dose-dependent increments in blood pressure and heart rate; plasma PRL was increased twofold after the higher dose of TRH. 4-F-TRH had effects similar to those of TRH on both the cardiovascular and PRL response. In contrast, the 2-TFM-TRH was significantly less active than TRH or 4-F-TRH in eliciting tachycardia, yet was noticeably more potent in affecting PRL release. These data suggest that the receptors for TRH-induced PRL release may be different from TRH-receptors which mediate central cardiovascular responses.     

Further studies on the mechanism of the cardiovascular effect of neurotensin in the rat

In vagotomized, pentobarbital-anesthetized rats, neurotensin (NT) (0.9 μg kg⁻¹) evokes biphasic pressor-depressor responses, the depressor component being the most important in magnitude. To support our previous proposal on the participation of mast cell mediators such as histamine and 5-hydroxytryptamine to the depressor effect of NT in rats, we studied the influence of various antiallergic and/or antiinflammatory drugs on the changes of blood pressure induced by NT and control agonists. The results showed that mepyramine and methysergide, given separately, inhibit by 54% and 34% respectively, the hypotensive phase of the response to NT. When given concomitantly, the two drugs convert the hypotensive phase of the response to NT, into a slight pressor effect. Disodium cromoglycate, an inhibitor of mast cell secretion, reduces by 96%, and dexamethasone by 100%, the depressor phase of the response to NT. These results clearly suggest the participation of mast cell histamine and 5-hydroxytryptamine in the hypotensive effect of NT in rats. To further our understanding of the mechanism by which NT and its mediators produce hypotension in rats we administered pentolinium, a ganglion-blocking drug, to vagotomized, pentobarbital-anesthetized rats. The results showed that pentolinium markedly inhibits the depressor components of the cardiovascular responses to NT, 5-hydroxytryptamine and histamine, and leads to the appearance of pressor effects for the three substances. These results were taken as an indication that NT and/or its mediators histamine and 5-hydroxytryptamine produce part of their hypotensive effect in rats by reducing the activity of the sympathetic nervous system.     

Influence of various drugs on the variations of blood pressure, hematocrit and plasma histamine caused by neurotensin and compound 48/80 in rats

Intravenous injections of neurotensin (NT) (0.5, 1 and 2 nmoles kg-1) evoked dose-dependent increases in histaminemia and hematocrit, and marked hypotensive effect, in anesthetized rats. The increase of plasma histamine was rapid in onset (within sec), peak plasma histamine being reached in less than 2 min. The decline of plasma histamine was gradual and almost complete 15 min after injection of NT. The hematocrit increased slowly, maximum values being obtained 5-10 min after injection of NT, and it persisted throughout the period of observation. The hypotensive effect of NT was rapid in onset and of prolonged duration. Compound 48/80, a well known histamine liberator and mast cell depletor, produced variations of blood pressure, of hematocrit and of plasma histamine very similar to those elicited by NT. Pretreatment of rats with cromoglycate, a well known mast cell stabilizer, or with dexamethasone, inhibited markedly the changes of histaminemia, of hematocrit and of blood pressure evoked by NT and compound 48/80. The results clearly suggest that the effects of NT on blood pressure and on vascular permeability in rats are mediated to some extent by mast cell histamine. Hexamethonium, a ganglion blocker, inhibited slightly the effect of NT on histaminemia but it did not block NT-induced changes of hematocrit. However, the hypotensive effect of NT was severely blocked in hexamethonium-treated rats. These results were interpretated as an indication that hexamethonium prevents NT-induced hypotension not merely by reducing the mobilization of mast cell histamine by NT but most likely by interfering with the mechanism by which NT and/or its mast cell mediators produce their effects on blood pressure.     

The effects of somatostatin, its fragments and an analog on electroconvulsive shock-induced amnesia in rats

In the present study the effects of intracerebroventricularly [icv] administered somatostatin [linear and cyclic], somatostatin3-6, somatostatin7-10 and des AA1,2,4,5,12,13 [D-Trp8] somatostatin [ODT8-SS] were investigated on electroconvulsive shock [ECS]-induced retrograde amnesia in rats. The ECS significantly decreased the foot shock-induced avoidance latency, and thus caused retrograde amnesia. Somatostatin [linear and cyclic] in a dose of 0.6 nM had no action on the ECS-induced retrograde amnesia, while in doses of 3 nM and [cyclic only] 6 nM it significantly prevented it. Somatostatin3-6, somatostatin7-10 and ODT8-SS in doses of 0.6, 3 and 6 nM had no effect on the ECS-induced amnesia. These results indicate that the whole sequence of the original somatostatin molecule is needed to block the ECS-caused retrograde amnesia.     

The role of central dopaminergic systems in the behavioral effects of H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH

In the present study the role of the central dopaminergic systems in the behavioral action of H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH was investigated. The heptapeptide inhibited the extinction of active avoidance behavior if the treatment was performed intracerebroventricularly (icv) in a dose of 1 microgram, but was ineffective in a dose of 0.1 micrograms. If the peptide was injected into the nucleus accumbens septi (NAS) in a dose of 1 microgram, 0.1 micrograms or 0.01 micrograms it inhibited the extinction, but in the latter dose its effect was only a short one. H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH in a dose of 1 microgram, 10 micrograms or 20 micrograms (icv) did not influence the turning activity of unilateral substantia nigra (USN)-lesioned animals. These results suggest that the NAS plays an important role in the behavioral action of H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH and the heptapeptide has no direct dopamine receptor stimulatory or dopamine-releasing effect in the striatum.     

Differences in the behavioral effects of single-stage and serial lesions of the hippocampus

This study compared the behavior of rats with serial destruction of the hippocampus in different topographical configurations and temporal sequences, to that of appropriate one-stage control groups. Animals that had partial bilateral damage at each operation were superior to one-stage controls in passive avoidance and reversal learning. Animals that had unilateral lesions at the first stage of surgery, followed by destruction in the contralateral hippocampus at the second stage, differed from controls in passive avoidance behavior only. In alternation tests, performance of serial and one-stage groups was statistically indistinguishable. One-stage operated animals allowed different lengths of postoperative convalescence prior to testing differed only in the spontaneous alternation tests. Implications of these results for hypotheses about serial recovery mechanisms and concepts of brain function are discussed.     

Analysis of the biphasic depressor-pressor effect and tachycardia caused by neurotensin in ganglion-blocked rats

In pentobarbital-anesthetized, vagotomized, ganglion-blocked rats, intravenous injections of NT (1–10 nmoles kg^?1) evoked biphasic depressor-pressor responses. The depressor component of the response was found to predominate when 1 nmole kg^?1 of NT was used, but to disappear almost completely with higher doses of the peptide. In contrast, the pressor component was more prominent with the use of higher doses of NT. Marked tachycardia usually accompanied the pressor effects of NT while little or no change of heart rate occurred during the depressor phase of the response. The depressor component of the response to 1 nmole kg^?1 of NT was markedly attenuated by captopril and by nephrectomy. This result suggests that depressor responses to NT in ganglion-blocked rats are somehow linked to the activity of the renin-angiotensin system. The pressor component of the responses to NT (4 nmoles kg^?1) and the associated tachycardia were reduced by 44% and 60% respectively, after ablation of adrenal glands. This result suggests that NT produces part of its cardiovascular effects in ganglion-blocked rats by stimulating the release of catecholamines from the adrenal glands. Prazosin, an alpha adrenergic receptor blocking drug, inhibited by 48% both the pressor effect and tachycardia evoked by NT while propranolol, a beta adrenergic receptor blocking drug, reduced by 67% the chronotropic effect of NT and only by 30% the pressor effect of the peptide. These results provide further evidence for the participation of catecholamines in the pressor effect and tachycardia evoked by NT in ganglion blocked rats. It also suggests that adrenal glands are the main source of the catecholamines involved in the pressor and chronotropic effect of NT. The partial blockade of the pressor effect and tachycardia of NT by low doses (1 mg kg^?1) of methysergide, a serotonin receptor antagonist, suggests that serotonin and/or their receptors may also contribute to the cardiovascular effects of NT in ganglion-blocked rats. When higher doses of methysergide producing blockade of both alpha adrenergic and serotonin receptors were used, NT evoked residual pressor effects (27%) preceded by significant depressor effects. The depressor responses were resistant to blockade by effective doses of atropine, propranolol, mepyramine, cimetidine, indomethacin and disodium cromoglycate, but it were markedly attenuated by captopril, again suggesting a possible link between the depressor activity of NT and the activity of the renin-angiotensin system in ganglion-blocked rats.     

Influence of histamine and prostaglandin on desensitization to neurotensin in rat blood pressure

Triphasic depressor-pressor-depressor blood pressure responses to neurotensin (NT: 1.67 μg/kg i.v.) in anesthetized rats were not elicited when the second dose of NT was administered 20 min after the first injection. Pretreatment of animals with histamine markedly reduced the depressor response to NT, and vice versa. The triphasic blood pressure pattern remained unaffected with acetylcholine and serotonin treatment, and hypotensive effects of acetylcholine and serotonin were not modified by NT. Attenuation of depressor response induced by the second injection of NT was antagonized by pretreatment with prostaglandin synthesis inhibitors such as indomethacin, mefenamic acid and acetylsalicylic acid. These results suggest that histamine and prostaglandins play a role in the development of desensitization to NT in rat blood pressure.     

Characterization of the inhibitory effect of [D-Trp11]-NT toward some biological actions of neurotensin in rats

We assessed the influence of various doses of [D-Trp11]-NT on the increase of histaminemia and hematocrit, and decrease of blood pressure, caused by intravenous injections of neurotensin (NT), substance P (SP) and compound 48/80 (C48/80) in anesthetized rats. [D-Trp11]-NT was found to inhibit dose-dependently and selectively the changes of histaminemia, hematocrit and blood pressure caused by NT. Since the highest dose of [D-Trp11]-NT utilized exhibited slight NT-like activity, we tested the possibility that desensitization rather than true pharmacological antagonism was responsible for the inhibitory action of [D-Trp11]-NT toward NT. This hypothesis was verified by evaluating the influence of intravenous infusions of sub-stimulatory and slightly active doses of NT on NT-induced effects. The sub-stimulatory dose (0.1 nmoles kg-1 min-1) as well as a higher dose rate (0.2 nmole kg-1 min-1) of NT were found to inhibit markedly the changes of histaminemia, hematocrit and blood pressure evoked by bolus doses of NT, without altering the effects of C48/80 on the same parameters. These results suggest that the inhibitory action of [D-Trp11]-NT toward NT-induced changes of histaminemia, hematocrit and blood pressure could be the result of receptor desensitization rather than to a true pharmacological antagonism. The results also suggest that the sensitivity of target tissues to exogenous NT could be modulated to some extent by endogenous circulating levels of NT.     

Presence of met-enkephalin in a somatostatin-synthesizing cell line derived from the fetal mouse hypothalamus

Immunocytochemical observations with purified antibodies revealed the presence of met-enkephalin-like material in the primitive nerve cell line F7 derived from the fetal mouse hypothalamus and previously shown to synthesize somatostatin (1). Radioimmunoassays associated with Bio-gel P₂ chromatography confirmed that met-enkephalin itself accounted (at least partly) for the positive immunocytochemical reaction. Trypsinization (± carboxypeptidase B treatment) of cell extracts significantly increased their met-enkephalin-like immunoreactivity therefore suggesting that met-enkephalin precursors were also present in the F7 clone. Parallel studies on the hypothalamus of fetal mice indicated that met-enkephalin but apparently no precursor was already present at embryonic day 15.The clonal cells F7 may be an appropriate model for investigating the functional correlate of the co-occurrence of metenkephalin and somatostatin in the same cells.     

Radiohistochemical localization of insulin receptors in the adult and developing rat brain

The light microscopic localization of insulin receptors in the rat central nervous system has been investigated by means of a new autoradiographic technique. These receptors are fairly evenly distributed in the adult brain except in the olfactory bulb. There, the external plexiform layer is especially rich in them. Insulin binding sites were more heterogeneously distributed in the fetal brain and exhibited a waxing and waning during development. Highest densities were observed at 15 days of gestation.     

An HPLC and RIA analysis of the cholecystokinin peptides in rat brain

Methanol and boiling water extracts of rat brain were chromatographed on two HPLC columns and the major cholecystokinin (CCK) peptides that eluted were detected by radioimmunoassay. The major CCK-like substance in both extracts co-chromatographed with CCK octapeptide sulfate on both columns, in agreement with previous reports of gel filtration chromatography of extracts of rat (1), porcine (2), human (2), and guinea pig brain (3). Minor immunoreactive components were detected in both extracts which chromatographed with CCK8 desulfate, CCK4 or 5, and variable amounts of partially or completely oxidized CCK8 sulfate and desulfate. Little or no immunoreactivity comigrated with sulfated or desulfated gastrin 17.In boiling water extracts, two additional peaks of immunoreactivity were detected, one which co-elutes with CCK33 and one which precedes CCK33 and has an apparent molecular weight of 40,000–50,000. This “pro-CCK” is not dissociated by treatment with sodium dodecyl sulfate and is considerably less hydrophobic than CCK8 or CCK4. It may be the same as the putative CCK precursor reported by Rehfeld to incorporate ³⁵S methionine in rat cortex (4) which yields CCK8 upon trypsinization (5).     

Distribution of cholecystokinin (CCK) in the hypothalamus and limbic system of the rat

Cholecystokinin (CCK) concentrations were determined in microdissected individual nuclei of the hypothalamus and limbic system using a specific and sensitive CCK radioimmunoassay (RIA). The CCK levels in the hypothalamus were highest (> 2 mg CCK8 sulfate equivalents/mg protein) in the ventromedial, dorsomedial, periventricular, arcuate nuclei, the median eminence, and mamillary body. Most regions of the limbic system had higher levels of CCK than the hypothalamus and in particular the CA3 region of the hippocampus, the lateral septal nucleus and the medial and lateral amygdaloid nuclei had levels of CCK which were similar to some cerebral cortical areas.This study confirms previous preliminary CCK distribution studies in the rat and indicates that CCK in addition to its wide distribution in the cerebral cortex, is present in abundance in many areas of the hypothalamus and limbic system.     

Normal CSF levels of met-enkephalin-like material in a case of naloxone—Reversible congenital insensitivity to pain

In a case of naloxone-reversible congenital insensitivity to pain, met-enkephalin-like immunoreactivity in the CSF was in the normal range and not affected by the administration of naloxone. Chromatographic analysis of the met-enkephalin-like material revealed that it corresponded to at least two classes of molecules. A clear difference in the relative proportions of these two classes was detected in the CSF of the patient insensitive to pain when compared to controls. The possible functional significance of this alteration is discussed in relation to the well known antinociceptive action of enkephalins.     

Changes in substance P and somatostatin in the spinal cord after traumatic spinal injury in the rat

Immunoreactive substance P (SPI) and somatostatin (SOMI) are found in spinal cord but their physiological roles remain speculative. Several classes of neuropeptides, including endogenous opioids and thyrotropin-releasing hormone (TRH), have been implicated in the pathogenesis or recovery from spinal cord injury. In the present studies, changes in SPI and SOMI were examined in the spinal cord after traumatic injury in the rat. Both peptides showed time-dependent, localized decreases at the injury site, which were statistically related to the degree of post-traumatic neurological dysfunction. Such changes differ from those of a number of other peptides after spinal injury and suggest that substance P and/or somatostatin may play a role in the secondary pathophysiological responses which follow trauma to the spinal cord.     

PHI stimulates intestinal fluid secretion

Regulatory peptides are likely to have a role in the control of net intestinal fluid transport. PHI is a peptide recently isolated from porcine duodenum which has been shown to have sequence homologies with other peptides of the glucagon-secretin family. We have studied the effect of intravenous infusion of synthetic PHI on net intestinal fluid transport in the rat small intestine. During PHI infusion net absorption was reduced in the duodenum and jejunum and net secretion was observed in the ileum. Thus synthetic PHI appears to be capable of strongly stimulating intestinal secretion in the rat.     

Pathways of TRH degradation in rat brain

We have investigated the TRH degradative enzymes in brain by examining the pattern of metabolites formed . The homogenate and three subcellular fractions of rat brain were separately incubated for 1, 5, and 15 minutes with ³H-TRH (pyro-glu-his-³H-proNH₂) at 37°C. TRH and its metabolites were separated on silica gel thin-layer chromatography plates. The crude homogenate and subcellular fractions each produced a characteristic pattern of metabolism. The homogenate metabolized TRH to TRH-OH, proline, and prolineamide. With the P₁ fraction, prolineamide and proline were the major metabolites. In both the homogenate and P₁ fraction incubations, histidyl-prolineamide appeared as a minor component. The P₂ fraction produced prolineamide and histidyl-prolineamide as the major metabolites while the cytosol metabolized TRH primarily to TRH-OH. Proline was formed during incubation with both cytosol and P₂ fractions.The TRH deamidase is found in the soluble fraction of brain tissue homogenate while the pyroglutamate aminopeptidase and the prolineamide cleaving enzyme are associated with particulate fractions. Histidyl-prolineamide is further degraded in the homogenate and P₁ fractions by a secondary metabolic pathway. Proline salvaging enzymes are present in all subcellular fractions of rat brain.     

Hypotensive effects of centrally and peripherally administered neurotensin and neurotensin derivatives in rats

We have evaluated and compared the hypotensive effects of intravenously (iv) and intracerebroventricularly (icv) injected NT, NT fragments and analogues in pentobarbital-anesthetized rats. The removal of the sequence pGlu¹-Leu²-Tyr³-Glu⁴-Asn⁵-Lys⁶-Pro⁷ reduced only slightly the hypotensive activity of NT injected iv and icv while the deletion of Leu¹³ or of Tyr¹¹-Ile¹²-Leu¹³-OH brought about a large decrease of potency (NT (1–12)) or a complete loss of hypotensive activity (NT (1–10)). The replacement of Tyr¹¹ with Trp produced a minor increase (～8%) or decrease (15%) of the potency of NT when injected iv and icv respectively. NT analogues in which Tyr¹¹ was replaced with Phe, D-Phe or D-Tyr exhibited large decreases of potency when injected iv but only small reductions of potency when injected icv. [D-Trp¹¹]-NT and [Tyr(Me)¹¹]-NT were relatively weak agonists using both routes of injection. Our results indicate that the chemical groups responsible for the hypotensive effect of peripherally and centrally injected NT are located in the same sequence (e.g. Arg⁹-Pro¹⁰-Tyr¹¹-Ile¹²-Leu¹³-OH). The results also suggest that the amino acid Tyr¹¹ contributes to a large extent to the hypotensive activity of peripherally injected NT. The importance of Tyr¹¹ for the hypotensive activity of centrally administered NT remains difficult to evaluate possibly because the receptors which subserve the hypotensive effect of centrally administered NT and/or the mechanisms responsible for the inactivation of NT in the brain and in the periphery, are different.     

Mixed opioid/nonopioid effects of dynorphin and dynorphin related peptides after their intrathecal injection in rats

Dynorphin dose-dependently increased the tail flick latency of rats to radiant heat following its intrathecal injection. This effect was accompanied by an alteration in motor function that was characterized by a flaccid extension of the hindlimbs and flaccidity of the tail. Naloxone (10 but not 1 mg/kg) blocked the antinociceptive effect and motor disturbance produced by dynorphin. The non-opioid analogue des-Tyr1-dynorphin(1-13) also increased tail flick latency and produced paralysis. Dynorphin(1-8) significantly elevated tail flick latency without affecting motor function. Furthermore, the effect of dynorphin(1-8) was blocked by 1 mg/kg naloxone. These data suggest a possible physiological role of dynorphin in influencing motor function in the spinal cord and a role of dynorphin(1-8) in modulating pain transmission. Another finding of the present study was that dynorphin was approximately ten times more potent in producing its effects when injected one day after surgery compared to when it when it was injected one week or more after surgery.     

The influence of oxytocin on the steady-state level and accumulation of serotonin in rat brain regions

The effects of graded amounts of centrally injected /intracerebroventricular, i.c.v./ oxytocin /OXT/ have been studied on the steady-state level and pargyline-induced accumulation of serotonin /5-HT/ in the hypothalamus, mesencephalon and dorsal hippocampus of rats. OXT only weakly affected the steady-state level of 5-HT. The pargyline-induced accumulation, on the other hand, was inhibited by central OXT treatment. Whereas OXT inhibited 5-HT accumulation in a dose-dependent manner in the hypothalamus, only the lowest amount of OXT was effective in the mesencephalon and the dorsal hippocampus. The data raise the possibility that an altered 5-HT metabolism is likely to have functional significance in the endocrine and behavioral effects of OXT.