Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract

BACKGROUND & AIMS: The gastrointestinal (GI) tract is a major source and target of somatostatin (SRIF). Recently, five pharmacologically different SRIF receptors (sst1-5) were cloned. The cellular and tissue distribution of the sst1-5 messenger RNAs (mRNAs) were studied in the rat GI tract using in situ hybridization histochemistry (ISHH). METHODS: Two sets of (35)S-uridine triphosphate (UTP)-labeled antisense and sense riboprobes were prepared for each sst. ISHH was conducted on frozen tissue samples from rat stomach, duodenum, jejunum, ileum, colon, and pancreas. RESULTS: mRNAs of all five sst-s are widely expressed in the rat GI tract. The distribution pattern for each sst mRNA was identical with both antisense probes. No specific signal was found with any of the sense probes. Each layer of the different parts of the gut expressed mRNAs of multiple sst subtypes. All organs expressed sst3 mRNA very intensely. The lowest levels of mRNA expression for all five subtypes within the GI tract were found in the pancreas. CONCLUSIONS: The widespread expression of sst mRNAs suggests a significant role for SRIF in the regulation of GI function.     

Changes of mu opioid receptor binding sites in rat brain following electroacupuncture

With [3H]-ohmefentanyl as a ligand, autoradiographic technique was used to observe the effect of electroacupuncture (EA) on mu opioid receptor binding sites in the brain areas related to pain modulation. The results were as follows: (1) The distribution of the mu receptor in the rat central nervous system was consistent in general with the results reported previously. (2) After EA of Tsu-San-Li, the mu receptor binding sites were increased significantly in the following examined structures: the caudate nucleus, septal nucleus, medial preoptic area, amygdalaoid nucleus, periaqueducal gray, interpeduncular nucleus, nucleus raphe magnus, and cervical and lumbar enlargements. The results indicate that EA is able to increase mu binding sites in the brain areas related to analgesia, suggesting the enhancement of mu receptor function by EA.     

Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat

We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis.     

Different patterns of induction of FGF-2, FGF-1 and BDNF mRNAs during kindling epileptogenesis in the rat

Neurotrophic factors (NTF) play important roles in the developing and in the adult brain. NTF involvement in neuronal plasticity is suggested by the modulation of NTF expression patterns in different physiological and pathological situations and by the effects they produce in the adult brain (e.g. axonal sprouting induction and neuroprotection). We used the RNAase protection assay to investigate the expression patterns of some NTFs during amygdala kindling, an animal model of epilepsy in which 'pathological' neuronal plasticity appears to occur. After a single kindling stimulation, fibroblast growth factor-2 (FGF-2) mRNA levels were increased in the hippocampus, the cortex and the hypothalamus, whereas they were not significantly altered in the thalamus and the striatum. A single stimulation did not alter fibroblast growth factor-1 (FGF-1) and brain-derived neurotrophic factor (BDNF) gene expression. Fully kindled animals, left unstimulated for a week, did not exhibit any alteration in the mRNA levels for any of the NTFs examined. However, in contrast with the effect of a single stimulation, amygdala stimulation of kindled animals (evoking a generalized tonic-clonic seizure) produced a great increase in hippocampal and cortical BDNF mRNA levels, but FGF-1 mRNA levels were not altered, and FGF-2 mRNA levels were significantly increased only in the cortex. These results suggest that different NTFs can be recruited at different stages of kindling epileptogenesis and, accordingly, may play different parts in the adaptive changes taking place in this experimental paradigm.     

Characterization and distribution of somatostatin SS-1 and SRIF-1 binding sites in rat brain: identity with SSTR-2 receptors

Somatostatin (SRIF) SS-1 binding sites were initially defined in radioligand binding studies performed in rat brain cerebral cortex membranes using [125I]204-090 (a radiolabelled Tyr3 analogue of SMS 201-995, octreotide). SRIF-1 recognition sites were defined in binding studies performed with [125I]MK 678 (seglitide). Both SS-1 and SRIF-1 sites were characterized by their high affinity for SRIF-14, SRIF-28 and for cyclic peptides such as octreotide and seglitide, in marked contrast to SS-2 and SRIF-2 sites which have very low affinity for these synthetic SRIF analogues. In the present study, SS-1 and SRIF-1 radioligand binding studies were performed in rat cortex membranes and compared to results obtained in cloned Chinese hamster ovary cells expressing human SSTR-2 receptors using [125I]204-090 and/or [125I]MK-678. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-1/SRIF-1 binding sites and recombinant SSTR-2 receptors were very similar and correlated highly significantly (r = 0.94-0.99); by contrast, correlation between SS-1 and SSTR-5 (r = 0.44) or SSTR-3 binding (r = 0.07) was not significant. Autoradiographic studies were performed in rat brain using both radioligands [125I]204-090 and [125I]MK-678 and compared with the distribution of SSTR-2 receptor mRNA determined using in situ hybridization. A clear overlap was observed between the distribution of SSTR-2 mRNA and binding sites labelled with both radioligands. SSTR-2 receptor-mediated inhibition of forskolin-stimulated adenylate cyclase in Chinese hamster ovary cells by a variety of SRIF analogues and short synthetic peptides displayed a rank order of potency highly similar to their rank order of affinity at SS-1/SRIF-1 binding sites. It is concluded that SS-1 and SRIF-1 binding sites respectively labelled with [125I]204-090 and [125I]MK 678, both display the pharmacological profile of SSTR-2 receptors, that the distribution of [125I]204-090 and [125I]MK-678 binding sites in rat brain is superimposable and largely comparable to that of SSTR-2 mRNA expression. It is also shown that neither [125I]204-090 nor [125I]MK-678 label SSTR-3 or SSTR-5 receptors in rat brain. Finally, it is demonstrated that SSTR-2 receptors can very efficiently couple to adenylate cyclase activity in an inhibitory manner.     

Distribution of thyrotrophin-releasing hormone receptor messenger RNA in rat pituitary and brain

X-ray diffraction methods have been used to determine the structure of the 8.3 kDa hydrophobic protein from soybean and to refine the atomic co-ordinates to a crystallographic R-factor of 18.7% at 1.8 A resolution. The molecule is a four-helix bundle, which together with the connecting loops and a twisted beta-strand form a spiral. The surface contains 70% apolar atoms, and the crystal packing is dominated by hydrophobic interactions, producing a two-dimensional sheet of protein molecules. Most of the 59 water molecules located are involved in hydrophilic contacts and their structural organization does not seem to be affected by the high hydrophobicity of the molecule. From the protein fold it appears that three of the four disulphide bridges are important for keeping the amino and carboxyl-terminal segments in place in the native form, while the central part of the molecule is stabilized by many hydrophobic interactions. Although the protein function is not known, a number of possibilities can be excluded on experimental grounds and by comparison with other members of the family.     

Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system

Glucagon-like peptide-1 (GLP-1) is derived from the peptide precursor pre-pro-glucagon (PPG) by enzymatic cleavage and acts via its receptor, glucagon-like peptide-1 receptor (GLP-1R). By using riboprobes complementary to PPG and GLP-1R, we described the distribution of PPG and GLP-1R messenger RNAs (mRNAs) in the central nervous system of the rat. PPG mRNA-expressing perikarya were restricted to the nucleus of the solitary tact or to the dorsal and ventral medulla and olfactory bulb. GLP-1R mRNA was detected in numerous brain regions, including the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus ceruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. These studies, in addition to describing the sites of GLP-1 and GLP-1R synthesis, suggest that the efferent connections from the nucleus of the solitary tract are more widespread than previously reported. Although the current role of GLP-1 in regulating neuronal physiology is not known, these studies provide detailed information about the sites of GLP-1 synthesis and potential sites of action, an important first step in evaluating the function of GLP-1 in the brain. The widespread distribution of GLP-1R mRNA-containing cells strongly suggests that GLP-1 not only functions as a satiety factor but also acts as a neurotransmitter or neuromodulator in anatomically and functionally distinct areas of the central nervous system.     

Specific reductions of striatal prodynorphin and D1 dopamine receptor messenger RNAs during cocaine abstinence

It is well established that the opioid neuropeptide and dopamine systems are altered following the use of cocaine. However very little information is available about their possible involvement during cocaine abstinence. In the present study, the mRNA expression of the dopamine receptors, D1 and D2, and the opioid peptides, prodynorphin and proenkephalin, were analyzed in the rat striatum using in situ hybridization histochemistry. Saline or cocaine (30 mg/kg, i.p.) were administered to rats once daily for 1 or 10 days. To examine cocaine abstinence, animals were treated for 10 days as described followed by a 10-day drug free period. Acute and intermittent cocaine administration elevated the prodynorphin mRNA expression in the dorsal striatum, consistent with previous reports, while the abstinent phase resulted in a significant reduction of prodynorphin mRNA levels in the ventrorostral striatum. The D1-receptor mRNA was decreased in the caudorostral striatum during cocaine withdrawal, a finding opposite to the increase observed following a single injection of the drug. Proenkephalin and the D2-receptor mRNAs were not altered during cocaine abstinence, though proenkephalin was elevated following acute but not repeated cocaine administration. These results show long-term suppression on prodynorphin and D1-receptor systems in specific striatal populations localized mainly in rostral areas during withdrawal from cocaine.     

The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain

The messenger RNA expression of non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4), considered alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid type, was investigated in rat brain by in situ hybridization histochemistry using oligonucleotide probes specific to each subunit sequence. GluR1-4 subunit messenger RNAs were expressed widely and abundantly throughout the CNS. However, the combination of expression pattern varied notably according to location. GluR2 messenger RNA was expressed most strongly and widely, with most areas except the Bergmann glia containing this messenger RNA. GluR4 messenger RNA was also present widely, although the expression level was low. However, we observed many areas which lacked or expressed very little GluR1 messenger RNA, such as some nuclei in the general motor system and auditory system. In addition, some nuclei in the hypothalamus and general somatosensory system lacked or expressed very little GluR3 messenger RNA. These results suggest that in the rat CNS non-N-methyl-D-aspartate receptors varied their composition according to the area where they were expressed, and that the combination pattern might be related to the functional role of neurons.     

Muscarinic cholinergic receptor binding in rat brain following traumatic brain injury

Recent evidence suggests that excessive activation of muscarinic cholinergic receptors (mAChRs) contributes significantly to the pathophysiological consequences of traumatic brain injury (TBI). To examine possible alterations in mAChRs after TBI, the affinity (Kd) and maximum number of binding sites (Bmax) of mAChRs in hippocampus, neocortex, brain stem and cerebellum were determined by [3H]QNB binding. Three groups of rats were examined: 1 h post-TBI (n = 21), 24 h post-TBI (n = 21) and sham-injured rats (n = 21). Kd values were significantly higher in hippocampus and brain stem at 1 but not 24 h post-TBI compared with sham-injured controls (P < 0.05). Kd values did not significantly differ in neocortex and cerebellum at 1 or 24 h post-TBI compared with sham-injured controls. Bmax values did not significantly differ in any brain areas at 1 or 24 h post-TBI compared with sham-injured controls. These results show that TBI significantly decreases the affinity of mAChRs in hippocampus and brain stem at an early stage post-TBI, which may contribute to desensitization of mAChRs after TBI. The findings of no change in Bmax values are consistent with a transient elevation in ACh concentrations after TBI.     

Diurnal variation of corticotropin-releasing factor binding sites in the rat brain and pituitary

OBJECTIVE: To discuss the usefulness of efficiency measures as instruments of monitoring and resource allocation by analyzing their invariance to changes in the operationalization of hospital production. STUDY SETTING: Norwegian hospitals over the three-year period 1989-1991. STUDY DESIGN: Efficiency is measured using Data Envelopment Analysis (DEA). The distribution of efficiency and the ranking of hospitals is compared across models using various distribution-free tests. DATA COLLECTION: Input and output data are collected by the Norwegian Central Bureau of Statistics. PRINCIPAL FINDINGS: The distribution of efficiency is found to be unaffected by changes in the specification of hospital output. Both the ranking of hospitals and the scale properties of the technology, however, are found to depend on the choice of output specification. CONCLUSION: Extreme care should be taken before resource allocation is based on DEA-type efficiency measures alone. Both the identification of efficient and inefficient hospitals and the cardinal measure of inefficiency will depend on the specification of output. Since the scale properties of the technology also vary with the specification of output, the search for an optimal hospital size may be futile.     

Suppression of kindling epileptogenesis in rats by intrahippocampal cholinergic grafts

The objective of our study was to investigate the effects of ultrasonic energy on tissues, using a porcine model, performed under various instrumental and procedural parameters. Domestic pigs were anesthetized and prepared for surgery. An incision was made on the side of the hip randomly assigned to the right or left side. Tumescence solution was infiltrated via a blunt tip, small diameter cannula, followed by performance of standard liposuction. On the contralateral side, a similar incision was made. For ultrasonic liposuction experiments without the sheath, a percutaneous introducer was inserted into the incision, which was protected at the entry site from contact with the cannula. Tumescence solution was infiltrated via a blunt tip, small diameter cannula, and then the site was treated with ultrasonic energy at maximum output from the machine with liposuction concurrent through the hollow cannula. The experiments with the sheath did not require a pretreatment with tumescence solution but consisted of tumescence solution pumped through the sheath at a low infusion rate, with concurrent treatment utilizing ultrasonically assisted liposuction through the central lumen of the cannula. In all cases, the lipoaspirate was preserved for biochemical analysis. After treatment, the pigs were euthanized, and samples for histopathology were taken. The pigs were then perfused with a radio-opaque solution through the left ventricle following preperfusion with saline. The groups were ultrasound-assisted liposuction with sheath (n = 3), ultrasound-assisted without sheath (n = 4), and tumescence alone (n = 1), with standard liposuction performed on the contralateral side for all ultrasound-assisted liposuction animals. The lipoaspirates from the ultrasonically assisted liposuction with the sheath showed significantly less blood loss (measured as hemoglobin in the aspirate) than standard liposuction (p = 0.012) at comparable levels of fat (measured as triglycerides in the aspirate). The lipoaspirates from ultrasound-assisted liposuction without the sheath showed blood loss comparable to that experienced with standard liposuction. The ratio of hemoglobin to triglyceride was lowest in the ultrasound-assisted group with (p = 0.01) and without (p = 0.06) the sheath when compared to traditional liposuction. In both of these treated groups, the radiograms of the perfused areas showed significantly less vascular disruption when compared with suction-assisted liposuction. Histopathologic examination of specimens taken from various treated areas showed substantial tissue damage comparable in ultrasound- and suction-assisted liposuction treated groups. This preliminary experimental study showed that ultrasound-assisted lipoplasty is comparable to traditional suction-assisted lipoplasty. Treatment with ultrasound provided more significant hemoglobin/triglyceride ratios, indicative of more lipid aspirated per hemoglobin lost, and better preservation of vascular tissues as demonstrated by our perfusion studies. Treatment with the sheath showed a significantly lower hemoglobin release with a diminished volume infused into the subcutaneous space during the procedure.     

Characterization of [3H]clozapine binding sites in rat brain

We examined the characteristics of [3H]clozapine binding sites in four rat brain regions (frontal cortex, limbic area, hippocampus and striatum) in order to elucidate the pharmacological profile of this unique atypical antipsychotic drug. The specific [3H]clozapine binding was found to be saturable and reversible in all these brain regions. Scatchard analysis of the saturation data indicated that the specific binding consisted of high- and low-affinity components. Displacement experiments showed that the muscarinic cholinergic receptor represented about 50% of [3H]clozapine binding in each brain area. Serotonin 5-HT2 and dopamine D4 receptor binding sites could also be detected by displacement experiments using ketanserin and nemonapride, respectively, in frontal cortex and limbic area, but not in hippocampus or striatum. Alpha-1, alpha-2, histamine H1, dopamine D1, D2, or D3 receptor components could not be determined within the high-affinity [3H]clozapine binding sites in any brain region. It is possible that the atypical property of clozapine may depend on the modulatory effect on dopaminergic function via 5-HT2 receptor blockade and/or may be mediated via D4 receptor blockade in the mesocortical and mesolimbic area.     

Neurotoxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels

Purified and reconstituted sodium channels have previously been shown to be functional in voltage-dependent ion conductance and in high affinity binding of tetrodotoxin and saxitoxin at neurotoxin receptor site 1 and alpha-scorpion toxins at receptor site 3, but high affinity binding of neurotoxins at receptor sites 2, 4, and 5 has not been demonstrated. The pyrethroid insecticide RU39568 enhances the specific binding of [3H]batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 on purified and reconstituted sodium channels up to 500-fold, reducing the Kd to 1.5 nM. Brevetoxins and alpha-scorpion toxins cause further allosteric enhancement of BTX-B binding. The pyrethroids deltamethrin and bifenthrin and the nonpyrethroid insecticide 2,2-bis(p-chlorophenyl)trichloroethane can partially substitute for RU39568 in enhancing BTX-B binding, but other pyrethroids are inactive. The brevetoxin PbTx-1 binds specifically to neurotoxin receptor site 5 on purified and reconstituted sodium channels with a Kd value of approximately 30 nM. Brevetoxin binding is enhanced up to 2-fold by the combination of batrachotoxin and RU39568. The allosteric enhancement of BTX-B binding by RU39568 is voltage dependent, decreasing progressively with depolarization to 0 mV. In contrast, PbTx-1 binding is not voltage dependent and PbTx-1 reduces the voltage dependence of the effect of RU39568. The results demonstrate restoration of high affinity binding and allosteric interactions of ligands at neurotoxin receptor sites 2 and 5 on purified and reconstituted sodium channels and provide an experimental approach to covalent labeling and identification of the peptide components of those receptor sites.     

Exposure to prenatal nicotine transiently increases neuronal nicotinic receptor subunit alpha7, alpha4 and beta2 messenger RNAs in the postnatal rat brain

This study determined the effects of prenatal nicotine exposure (2 mg/kg/day) in Sprague Dawley CD rats via subcutaneously implanted osmotic minipumps, during gestational days 7-21, on postnatal levels of neuronal nicotinic receptor alpha4, alpha7 and beta2 subunit messenger RNAs. Northern analysis of postnatal day 1, 7, 14 and 28 hippocampal/septal and cortical total RNA using alpha-[32P]dCTP-labeled alpha4, alpha7 and beta2 complementary DNA probes identified a single (5.7-kb) alpha7 messenger RNA, three (2.4-, 3.8- and 8.0-kb) alpha4 messenger RNAs and four (3.7-, 5.0-, 7.5- and 10.0-kb) beta2 messenger RNAs. In comparison to prenatal saline, prenatal nicotine produced several significantly higher messenger RNA levels (cortical: 5.7-kb alpha7, 2.4-, 3.8- and 8.0-kb alpha4, 10.0-kb beta2; hippocampal/septal: 2.4- and 8.0-kb alpha4); these increases occurred predominantly on, but were not restricted to, postnatal day 14. Effects of nicotine were generally resolved by postnatal day 28. Collapsing the data across sex and age, a significant treatment effect indicated that hippocampal/septal and cortical 8.0-kb alpha4 messenger RNA levels and 10.0-kb beta2 messenger RNA levels were significantly higher following prenatal nicotine exposure. This is the first study indicating that prenatal nicotine produces alterations in developing postnatal rat neuronal nicotinic receptor messenger RNA levels, possibly by premature stimulation of neuronal nicotinic receptors. These results further implicate the teratogenic potential of nicotine in postnatal neuronal development.     

Cytochemical studies of lectin binding sites in smooth membrane cisternae of rat brain

The cytochemical localization of concanavalin A (con A) binding sites has been studied in Purkinje cell axons and presynaptic terminals of rat cerebellum. Smooth membrane cisternae just beneath the axolemma contain con A binding sites on the side of the membrane facing the cisternal space. At certain regions, such as the node of Ranvier, these cisternae lie in virtual apposition to the axolemma. Such a specialized system of cisternae could serve as a channel through which some of the materials synthesized in the Purkinje somata are moved to the axon terminals. The close association of the cisternae and axolemma at certain regions could be a site at which some of the transported materials contribute to renewal of the axolemma. The con A binding sites on intracellular membranes of the Purkinje cell are removed by prior glycosidic and proteolytic enzyme digestions. The results suggest that at least some of the carbohydrates lining membrane cisternae are glycoprotein in nature.     

Distinct changes in peptide YY binding to, and mRNA levels of, Y1 and Y2 receptors in the rat hippocampus associated with kindling epileptogenesis

Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of 125I-peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% (p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham-stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36-87% (p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50-64% (p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% (p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y-mediated neurotransmission.     

Chronic antipsychotic treatment alters glycine-stimulated NMDA receptor binding in rat brain

A growing body of studies have confirmed that autoantibodies against beta 1-adrenoceptors are present in different types of cardiomyopathy. This suggests that they play a role in the pathophysiology of the disease. This article will review the data indicating the presence of anti-beta 1-adrenoceptor autoantibodies in cardiomyopathy. It will focus upon their structural and functional properties which could explain their possible role in the induction and development of cardiomyopathic diseases.