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PubMed1996

Permanent inhibition of angiotensinogen synthesis by antisense RNA expression.

The renin-angiotensin system plays a pivotal role in blood pressure regulation. Recent molecular biological findings led to the new concept that in addition to the classic endocrine system, local tissue systems may also play an important role in cardiovascular diseases such as hypertension. In particular, the brain renin-angiotensin system was shown to influence the central control of blood pressure and is thought to contribute to the hypertensive phenotype of genetically hypertensive rat models. To identify the physiological role of these local systems, we established an antisense strategy to downregulate the expression of the precursor hormone angiotensinogen (AOGEN) in cell culture, which can also be used to establish transgenic rat lines. Plasmids encoding an RNA sequence complementary to the rat AOGEN mRNA under control of different viral and tissue-specific promoters were constructed and transfected into an AOGEN-expressing cell line. A competitive reverse transcription-polymerase chain reaction method was established for the quantification of AOGEN mRNA. Depending on the level of antisense RNA, the expression of the AOGEN gene was reduced down to 22% of control levels. Furthermore, the secretion of AOGEN protein was totally abolished. These results clearly demonstrate that the antisense constructs used are functional in reducing the AOGEN gene expression in vivo and can be used for the production of transgenic rats.

Angiotensinogen↗
PubMed1996

Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels.

Outward rectifier K+ channels have a characteristic structure with six transmembrane segments and one pore region. A new member of this family of transmembrane proteins has been cloned and called Kv8.1. Kv8.1 is essentially present in the brain where it is located mainly in layers II, IV and VI of the cerebral cortex, in hippocampus, in CA1-CA4 pyramidal cell layer as well in granule cells of the dentate gyrus, in the granule cell layer and in the Purkinje cell layer of the cerebellum. The Kv8.1 gene is in the 8q22.3-8q24.1 region of the human genome. Although Kv8.1 has the hallmarks of functional subunits of outward rectifier K+ channels, injection of its cRNA in Xenopus oocytes does not produce K+ currents. However Kv8.1 abolishes the functional expression of members of the Kv2 and Kv3 subfamilies, suggesting that the functional role of Kv8.1 might be to inhibit the function of a particular class of outward rectifier K+ channel types. Immunoprecipitation studies have demonstrated that inhibition occurs by formation of heteropolymeric channels, and results obtained with Kv8.1 chimeras have indicated that association of Kv8.1 with other types of subunits is via its N-terminal domain.

Amino Acid Sequence↗
PubMed1997

A non-retroviral RNA virus persists in DNA form.

Infection of adult mice with lymphocytic choriomeningitis virus (LCMV), a non-cytopathic segmented RNA virus, leads initially to generalized infection, followed by clearance and subsequent life-long immunity. Indirect evidence has suggested that viral antigens may persist in lymphoid tissues during the phase of immunological memory, but viral genomic RNA has not been detected in previous studies. During a search for persistent virus in the spleen, we identified LCMV-specific sequences present as DNA by polymerase chain reaction (PCR) in mice over 200 days after infection. In vivo and in vitro studies revealed that reverse transcription of viral RNA into complementary DNA occurred after acute infection of cells of its natural hosts, mouse and hamster, but not of other species and could be inhibited in vitro by azidothymidine (AZT), indicating that this was mediated by endogenous reverse transcriptase activity. These findings reveal a surprising and new pathway of interaction between exogenous RNA viruses and endogenous retroviral, and perhaps other host components, that results in the persistence of virally determined DNA. We speculate that the latter may function in vivo as a form of DNA vaccine.

Animals↗
PubMed1972

In vitro synthesis of DNA complementary to purified rabbit globin mRNA (RNA-dependent DNA polymerase-reticulocyte-hemoglobin-density gradient centrifugation-oligo(dT) primer).

Several properties of the viral RNA-dependent DNA polymerases and of rabbit globin mRNA make it possible to consider synthesis of the globin gene in vitro. These enzymes copy an RNA template using a short sequence of complementary nucleotides as a primer. Furthermore, globin mRNA has a 3'-terminal sequence of adenylic acid residues that make it particularly suitable as a template, since oligo(dT) can be annealed to a specific site on the mRNA. This small primer could phase the DNA polymerase, possibly ensuring that replication is initiated from that end of the globin message. We have used this approach and find that purified mRNA is an efficient template for the polymerase enzyme. The reaction requires the RNA template and the four deoxyribonucleoside triphosphates, and it is markedly stimulated by the addition of oligo(dT). Consistent with the expectation that the oligo(dT) uniquely phases the polymerase at an adenine-rich region in the globin message, oligo(dG), oligo(dC), and oligo(dA) fail to serve as primers. The product has a density intermediate between that of DNA and RNA, and shifts to a lighter DNA density after treatment with base. Further, it is specifically complementary to globin mRNA and sediments slightly faster in an alkaline sucrose gradient than a DNA standard that has a molecular weight of 129,000. The data suggest that a major portion of the DNA product is a sequence of at least 500 bases, about 50 more than would be necessary to encode rabbit globin. The potential usefulness of this interesting product is discussed.

Animals↗
PubMed1996

The establishment of the hepatic architecture is a prerequisite for the development of a lobular pattern of gene expression.

We have studied the expression patterns of ammonia-metabolising enzymes and serum proteins in intrasplenically transplanted embryonic rat hepatocytes by in situ hybridisation and immunohistochemical analysis. The enzymic phenotype of individually settled hepatocytes was compared with that of hepatocytes being organised into a three-dimensional hepatic structure. Our results demonstrate that development towards the terminally differentiated state with zonal differences in enzyme content requires the incorporation of hepatocytes into lobular structures. Outside such an architectural context, phenotypic maturation becomes arrested and hepatocytes linger in the protodifferentiated state. These features identify the foetal period as a crucial time for normal liver development and show that the establishment of the terminally differentiated hepatocellular phenotype, beginning with the differentiation of hepatocytes from the embryonic foregut, is realised via a multistep process.

Albumins↗
PubMed1997

The sugar specificity of Na+/glucose cotransporter from rat jejunum.

A cDNA for a Na+/glucose cotransporter was cloned from rat jejunum cDNA library. This transporter was expressed in Xenopus oocytes by injection of cRNA synthesized from the cDNA, and the transporter ability was electrophysiologically examined. The cotransporter had a very narrow sugar specificity. Only D-glucose, D-galactose, and some of their derivatives elicited significant electrical responses. These results of sugar specificity were compared with those of the H+/hexose cotransporter of Chlorella. Dose-response relationships of several sugars followed a simple Michaelis-Menten type of kinetics. Both Vm and Km were dependent on the sugars. Not only the affinity of sugars to the cotransporter but also the rate of conformational change of the cotransporter loaded with the sugar and Na+, which translocates them from outside to inside, possibly depends on the sugar structure. The rate-limiting step of the transportation may be the conformational change, i.e., isomerization, of the cotransporter that translocates both the sugar and Na+ from outside to inside.

Animals↗
PubMed1976

Isolation and characterization of Sendai virus DI-RNAs.

When passaged at high multiplicity, four strains of Sendai virus all showed evidence that they contained defective interfering (DI) particles. RNA isolated from nucleocapsids of cells infected with the high multiplicity passage stocks was found to consist of only minor amounts of nondefective genome length RNA and major amounts of smaller RNAs, the DI-RNAs. These DI-RNAs were found to have unusual and variable sedimentation properties in sucrose gradients, but were found to represent unique segments of the viral genome by length measurements in the electron microscope and by hybridization. A striking feature of the DI-RNAs is their ability to form circular structures, indicating that the ends of the DI-RNA are complementary. The implications of this finding in terms of the mechanism of genome replication is discussed.

Cell Line↗
PubMedDate not supplied

Positive and negative strands of HCV-RNA in sera and peripheral blood mononuclear cells of chronically hemodialyzed patients.

BACKGROUND: Hepatitis C virus (HCV) RNA can be detected in sera and peripheral blood mononuclear cells (PBMC) of patients undergoing chronic hemodialysis. However, the natural course of HCV infection in this group of patients is not fully known. Although the exact mechanism of HCV replication is not completely explained, there is evidence that HCV replicate through synthesis of complementary negative (-) RNA strand, whereas positive (+) RNA strand serves as a template. Thus, the detection of negative HCV-RNA strand can be regarded as a marker of ongoing viral replication. MATERIAL AND METHODS: We investigated the prevalence of (+) and (-) strands of HCV-RNAs in sera and PBMC of 45 chronically hemodialyzed patients using PCR methods. We also determined HCV genotypes and their subtypes by Inno-LIPA method. RESULTS: Eight (17.8%) of analyzed patients were anti-HCV positive. In this group, we detected HCV-RNA (+) strands in sera and PBMC in 2 and 4 cases respectively, whereas HCV-RNA (-) ones were found in PBMC of 4 patients. Among the remaining 37 anti-HCV negative patients we found HCV-RNA positive strands in sera and PBMC in 2 and 3 cases respectively, whereas HCV-RNA negative strand was present in PBMC in one of them. CONCLUSIONS: Our results indicate that HCV actively replicate in PBMC in chronically hemodialyzed patients. In number of patients HCV-RNAs can be detected only in PBMC without concomitant presence of viremia or anti-HCV in sera. We did not find any correlation between genotypes of HCV and presence of HCV-RNAs strands in PBMC of the patients.

Adult↗
PubMed1999

Heteromeric assembly of Kv2.1 with Kv9.3: effect on the state dependence of inactivation.

Modulatory alpha-subunits of Kv channels remain electrically silent after homomeric expression. Their interactions with Kv2 alpha-subunits via the amino-terminal domain promote the assembly of heteromeric functional channels. The kinetic features of these heteromers differ from those of Kv2 homomers, suggesting a distinct role in electrical signaling. This study investigates biophysical properties of channels emerging from the coexpression of Kv2.1 with the modulatory alpha-subunit Kv9.3. Changes relative to homomeric Kv2.1 concern activation, deactivation, inactivation, and recovery from inactivation. A detailed description of Kv2.1/Kv9.3 inactivation is presented. Kv2.1/Kv9.3 heteromers inactivate in a fast and complete fashion from intermediate closed states, but in a slow and incomplete manner from open states. Intermediate closed states of channel gating can be approached through partial activation or deactivation, according to a proposed qualitative model. These transitions are rate-limiting for Kv2.1/Kv9.3 inactivation. Finally, based on the kinetic description, we propose a putative function for Kv2.1/Kv9.3 heteromers in rat heart.

Animals↗
PubMed2005

RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domains.

Members of the RIC-3 gene family are effectors of nicotinic acetylcholine receptor (nAChR) expression in vertebrates and invertebrates. In Caenorhabditis elegans RIC-3 is needed for functional expression of multiple nAChRs, including the DEG-3/DES-2 nAChR. Effects of RIC-3 on DEG-3/DES-2 functional expression are found in vivo and following heterologous expression in Xenopus leavis oocytes. We now show that in X. leavis oocytes RIC-3 also affects the kinetics and agonist affinity properties of the DEG-3/DES-2 receptor. Because these effects are mimicked by increasing the ratio of DEG-3 subunits within DEG-3/DES-2 receptors, this suggests that RIC-3 may preferentially promote maturation of DEG-3-rich receptors. Indeed, effects of RIC-3 on functional expression of DEG-3/DES-2 positively correlate with the DEG-3 to DES-2 ratio. All RIC-3 family members have two transmembrane domains followed by one or two coiled-coil domains. Here we show that the effects of RIC-3 on functional expression and on receptor properties are mediated by the transmembrane domains and do not require the coiled-coil domains. In agreement with this, mammals express a RIC-3 transcript lacking the coiled-coil domain that is capable of promoting DEG-3/DES-2 functional expression. Last, we show that RIC-3 affects DEG-3 quantity, suggesting stabilization of receptors or receptor intermediates by RIC-3. Together our results suggest that subunit-specific interactions of RIC-3 with nAChR subunits, mediated by the transmembrane domains, are sufficient for the effects of RIC-3 on nAChR quantity and quality.

Amino Acid Sequence↗
PubMed1992

Design of polymerase chain reaction primers for the selective amplification of HIV-1 RNA in the presence of HIV-1 DNA.

OBJECTIVE: To develop a sensitive method for the specific detection of HIV-1 RNA. DESIGN: Following reverse transcription, the presence of HIV-1 RNA can be detected by polymerase chain reaction (PCR) amplification. Since specific detection of HIV-1 RNA may be complicated by contamination with minute quantities of HIV-1 DNA, samples are treated with deoxyribonuclease (DNase) prior to analysis. This additional step increases the possibility of RNA degradation and sample contamination. METHODS: A primer, HG141, was designed to hybridize to the poly(A) tract present in HIV-1 genomic and all HIV-1 messenger (m) RNA with its 5' end and to the region upstream of the poly(A) tract with its 3' end. The increased stability of the HG141 primer/HIV-1 RNA or complementary (c) DNA complex, enabled PCR amplification to be performed with HG141 and the return primer HG62 at an annealing temperature above the melting temperature (Tm) of the primer-HIV-1 DNA complex. RESULTS: After reverse transcription of samples obtained from HIV-1-infected H9 cells, HG62/141-primed PCR amplification specifically detected HIV-1 RNA sequences without the need for DNAase pre-treatment. This technique was more sensitive for the detection of HIV-1 RNA than SK38/39-primed PCR amplification of DNase-treated samples. CONCLUSIONS: Since the presence of HIV-1 RNA is indicative of HIV-1 replication for the presence of HIV-1 virions, the RNA-specific primer described should facilitate the assessment of HIV-1 replication and the plasma HIV-1 viral load in HIV-1-infected individuals. This should prove useful in the evaluation of the effects of therapeutic interventions on HIV-1 infection.

Base Sequence↗
PubMed2005

Slicer function of Drosophila Argonautes and its involvement in RISC formation.

Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ("Slicer") activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here we show that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. We show that upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as "Slicer" of the passenger strand of the siRNA duplex.

Adenosine Triphosphate↗
PubMed1999

Psx homeobox gene is X-linked and specifically expressed in trophoblast cells of mouse placenta.

We previously isolated a cDNA clone for a homeobox-containing gene with its expression restricted to the extraembryonic tissues. In this study, Psx gene expression was further examined using in situ hybridization to determine the cellular distribution of Psx transcripts during embryo development. Psx expression was first detected at embryonic day 8.5 only in trophoblast giant cells and chorionic ectoderm. At E 9.5 and E 13.5, the expression was restricted to the giant cells and the labyrinthine trophoblast layer. In addition, the gene expression was detected in differentiated Rcho-1 trophoblast cells in vitro, which is typical of trophoblast giant cells in vivo, but not in proliferating Rcho-1 cells and HRP-1 cells. Interestingly, rat Psx homologue mRNA is about 200 bp shorter than mouse Psx, suggesting that there is a high degree of sequence divergence between the mouse and rat Psx homologues. The sequence divergence, perhaps as a result of rapid evolution, is further supported by the zoo blot analysis because the Psx gene was detectable only in mouse and rat but not in other vertebrate species tested. Psx is localized to the murine X chromosome. Taken together, our results suggest that Psx gene plays a unique role in the function of differentiated trophoblast cells and also serves as a useful model for studying trophoblast cell lineages and the rapid evolution of homeobox genes.

Amino Acid Sequence↗
PubMed2003

Ethanol enhances alpha 4 beta 3 delta and alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at low concentrations known to affect humans.

gamma-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (> or = 60 mM). We show that GABARs containing the delta-subunit, which are highly sensitive to gamma-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt/vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR delta-subunit, which is thought to be associated exclusively with alpha 4- and alpha 6-subunits in vivo, and the beta 3-subunit, which has recently been shown to be essential for the in vivo anesthetic actions of etomidate and propofol. GABARs containing beta 2-instead of beta 3-subunits in alpha 4 beta delta- and alpha 6 beta delta-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing gamma 2-instead of delta-subunits with alpha 4 beta and alpha 6 beta are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that "extrasynaptic" delta-subunit-containing GABARs, but not their "synaptic" gamma-subunit-containing counterparts, are primary targets for EtOH.

Alcohol Drinking↗
PubMed2004

Opposite effects of a single IIIS5 mutation on phenylalkylamine and dihydropyridine interaction with L-type Ca2+ channels.

Replacement of L-type Ca(2+) channel alpha(1) subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca(2+) channels eliminates high dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on phenylalkylamine interaction, we exploited the availability of Ca(v)1.2DHP(-/-) mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (-)-[(3)H]devapamil occurred to Ca(v)1.2DHP(-/-) mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (-)-verapamil but not for (-)-gallopamil. The mutation caused a pronounced slowing of (-)-[(3)H]devapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 mum (-)-verapamil inhibited a significantly larger fraction of Ba(2+) inward current through mutant than through WT channels. Our results provide evidence that phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca(2+) channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect phenylalkylamine and dihydropyridine binding to L-type Ca(2+) channels.

Animals↗
PubMed2004

Functional comparison of mouse slc26a6 anion exchanger with human SLC26A6 polypeptide variants: differences in anion selectivity, regulation, and electrogenicity.

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.

Animals↗
PubMed2006

Stimulation of the epithelial sodium channel (ENaC) by cAMP involves putative ERK phosphorylation sites in the C termini of the channel's beta- and gamma-subunit.

The mechanisms involved in the regulation of the epithelial sodium channel (ENaC) via the cAMP pathway are not yet completely understood. The aim of the present study was to investigate cAMP-mediated ENaC regulation in Xenopus laevis oocytes heterologously expressing the three subunits (alphabetagamma) of rat ENaC and to determine the ENaC regions important for mediating the stimulatory effect of cAMP. In oocytes treated for about 24 h with 1 mm 3-isobutyl-1-methylxanthine (IBMX) and 1 microm forskolin (FSK) so as to increase intracellular cAMP, the amiloride-sensitive whole cell current (DeltaI(Ami)) was on average 10-fold larger than DeltaI(Ami) in matched control oocytes. This effect on DeltaI(Ami) was paralleled by an increase in ENaC surface expression caused by a reduced rate of ENaC retrieval. In addition, IBMX/FSK also enhanced ENaC open probability from about 0.2 to 0.5. The stimulatory effect of IBMX/FSK was dependent on the presence of intact PY motifs in the C termini of the channel. Mutagenesis of putative protein kinase A and CK-2 consensus motifs in the cytosolic domains of the channel did not reveal critical sites involved in mediating the stimulatory effect of IBMX/FSK. In contrast, site-directed mutagenesis of two putative ERK-consensus motifs (T613A in betaENaC and T623A in gammaENaC) largely reduced the stimulatory effect of IBMX/FSK. Phosphorylation of these ERK sites has previously been reported to enhance the interaction of ENaC and Nedd4 (Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R., and Garty, H. (2002) J. Biol. Chem. 277, 13539-13547). Using co-expression experiments we demonstrated that mutating the two ERK sites attenuates the inhibitory effect of Nedd4-2 on ENaC currents. We conclude that an increase in intracellular cAMP favors the dephosphorylation of the two ERK sites, which reduces channel retrieval and increases P(O) by modulating ENaC/Nedd4 interaction. This defines a novel regulatory pathway likely to be relevant for cAMP-induced stimulation of ENaC in vivo.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Calcitonin gene-related peptide-containing neurons supplying the rat digestive system: differential distribution and expression pattern.

In the enteric nervous system, calcitonin gene-related peptide (CGRP) immunoreactivity is localized to a substantial number of capsaicin-sensitive afferent fibers and to intrinsic neurons and processes. CGRP immunoreactivity detected by immunohistochemistry represents the expression of two distinct genes, the calcitonin/alpha-CGRP and the beta-CGRP genes, which have different tissue distributions. In the present study, we used (1) in situ hybridization histochemistry and ribonucleic acid (RNA) blot hybridization with RNA probes complementary to the divergent sequences of alpha- and beta-CGRP messenger RNAs (mRNAs) to differentiate which CGRP gene was expressed in enteric and afferent neurons; and (2) axonal transport approaches in combination with CGRP immunohistochemistry to define the location of CGRP-containing afferent neurons supplying the digestive system. In situ hybridization histochemistry with [35S]-labeled RNA probes indicated that in the gastrointestinal tract beta-CGRP mRNA, but not alpha-CGRP mRNA, was expressed in enteric neurons confined to the myenteric and submucous plexuses of the small and large intestine. In dorsal root and vagal sensory ganglia, mRNAs for alpha-CGRP and beta-CGRP were both present in a vast population of neurons, with an overlapping pattern, even though the alpha-CGRP signal appeared more intense. RNA blot hybridization analysis showed a single band of hybridization at 1.2 Kb with the beta-CGRP RNA probe in RNA extracts from muscle layer-myenteric plexus and submucosal layer preparations of the ileum, and from dorsal root ganglia; it also showed a single band at 1.3 Kb with the alpha-CGRP RNA probe in extracts from dorsal root ganglia, but not from the intestine. These findings further support the differential expression of alpha- and beta-CGRP mRNAs. Retrograde transport of fast blue or fluorogold coupled with CGRP immunohistochemistry demonstrated that the vast majority of CGRP-containing afferent neurons supplying the stomach, proximal duodenum, and pancreas were located in dorsal root ganglia at the middle and lower thoracic and at the upper lumbar levels, and represented a major component of the afferent innervation of these viscera (up to 89%). Approximately 50% of CGRP-immunoreactive afferent neurons also expressed tachykinin (TK) immunoreactivity, as shown by triple labeling. Only a minor component of the afferent innervation of the stomach, duodenum, and pancreas derived from vagal CGRP-containing neurons (less than 8%). A large portion of these neurons (an average of 62%) also contained TK immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗