ABSTRACT
We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.
Subject(s)
Muscle, Smooth/physiology , Sodium Channels/metabolism , Uterus/physiology , Animals , Calcium/pharmacology , Epithelial Sodium Channel Agonists , Epithelial Sodium Channels/genetics , Epithelial Sodium Channels/metabolism , Female , Gene Expression , Muscle Contraction , Muscle, Smooth/drug effects , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Wistar , Sodium Channel Agonists , Sodium Channels/genetics , Uterus/drug effects , Veratridine/pharmacologyABSTRACT
Voltage-gated Na+ channels are composed of one alpha subunit and one or more auxiliary beta subunits. A reverse transcription-polymerase chain reaction assay was used to analyse the expression of the nine known alpha subunits (Na(v)1.1-Na(v)1.9) in 20 different human tissues. The mRNA expression of the currently known beta subunits (beta1, beta2, beta3 and beta4) was also assessed. The mRNAs of voltage-gated Na+ channel alpha and beta subunits were found in a wide variety of human tissues assayed and were present in neuronal and non-neuronal types of cells. These data suggest that, in addition to its well-established role in skeletal muscle, cardiac cells and neurons, voltage-gated Na+ channels might play important, still undetermined local roles in the regulation of cellular functions. These channels could emerge in the next future as potential, new therapeutic targets in the treatment of visceral diseases.
Subject(s)
Gene Expression Profiling , Nerve Tissue Proteins/genetics , RNA, Messenger/metabolism , Sodium Channels/genetics , Adult , Female , Fetus , Gene Expression Regulation, Developmental/genetics , Humans , Male , NAV1.1 Voltage-Gated Sodium Channel , Protein Subunits/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The tachykinins substance P, neurokinin A and neurokinin B are implicated in different diseases and play an important role in neuroimmunomodulation. These peptides interact with three distinct types of tachykinin receptors termed NK(1), NK(2) and NK(3). While most mammalian genes encoding G protein-coupling cell membrane receptors are intron-less, the three tachykinin receptors contain introns in their genomic structures. In the present study, we have identified a splice variant of the tachykinin NK(2) receptor that results from skipping of exon 2 in the processing of the tachykinin NK(2) receptor mRNA. By using reverse transcription-polymerase chain reaction analysis, we observed that the tachykinin NK(2) receptor splice variant, that we named NK(2)beta, appeared in different human and rat tissues that also express the wild type, tachykinin NK(2)alpha isoform. Compared to tachykinin receptor NK(2)alpha isoform mRNA levels, the NK(2)beta isoform was strongly expressed in human and rat uteri, expressed in a moderate degree in the rat urinary bladder, colon, duodenum and stomach and unexpressed in the rat cerebral cortex, kidney, thoracic aorta, skeletal muscle and heart. These data describe the first known tachykinin receptor splice variant and suggest that the variety of tachykinin receptors may be further expanded through the generation of splicing isoforms. The presence of the truncated isoform may have a physiological significance in the regulation of tachykinin NK(2) receptor protein levels.
