ABSTRACT
Smooth muscle myosin phosphatase dephosphorylates the regulatory myosin light chain and thus mediates smooth muscle relaxation. The activity of this myosin phosphatase is dependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1 have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle section of chicken and rat MYPT1 genes revealed that each gene gave rise to isoforms by cassette-type alternative splicing of exons. In chicken, a 123-nucleotide exon was included or excluded from the mature mRNA, whereas in rat two exons immediately downstream were alternative. MYPT1 isoforms lacking the alternative exon were only detected in mature chicken smooth muscle tissues that display phasic contractile properties, but the isoform ratios were variable. The patterns of expression of rat MYPT1 mRNA isoforms were more complex, with three major and two minor isoforms present in all smooth muscle tissues at varying stoichiometries. Isoform switching was identified in the developing chicken gizzard, in which the exon-skipped isoform replaced the exon-included isoform around the time of hatching. This isoform switch occurred after transitions in myosin heavy chain and myosin light chain (MLC(17)) isoforms and correlated with a severalfold increase in the rate of relaxation. The developmental switch of MYPT1 isoforms is a good model for determining the mechanisms and significance of alternative splicing in smooth muscle.
Subject(s)
Gene Expression Regulation, Developmental , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth/enzymology , Phosphoprotein Phosphatases/genetics , Animals , Aorta , Base Sequence , Chick Embryo , Chickens , Cloning, Molecular , Exons , Gene Expression Regulation, Enzymologic , Gizzard, Avian , Isoenzymes/genetics , Molecular Sequence Data , Muscle Development , Muscle, Smooth/embryology , Muscle, Smooth/growth & development , Muscle, Smooth, Vascular/embryology , Muscle, Smooth, Vascular/growth & development , Myosin-Light-Chain Phosphatase , Phenotype , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Sequence Alignment , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
Three experiments were conducted in unanesthetized rats made hypothyroid (Hypo) or maintained as euthyroid controls (Eu) to examine general cardiovascular responsiveness [experiment I (Exp I)]; responsiveness to a serotonin (5-HT2) agonist, dl-2,5-dimethoxy-4-iodoamphetamine [DOI intracerebroventricularly; experiment II (Exp II)]; or responsiveness to a 5-HT(1A) agonist dl-8-hydroxydipropyl-aminotetralin hydrobromide [8-OH-DPAT intracerebroventricularly; experiment III (Exp III)]. In Exp I, intravenous infusions of phenylephrine and nitroprusside provided little evidence that findings in Exp II and III were caused by generalized impairment in cardiovascular responsiveness in Hypo. In Exp II and III, Eu and Hypo were given either intra-arterial atropine or vehicle. Atropine significantly elevated heart rate (Exp II and III) and mean arterial pressure (Exp II) in Eu only. When compared with Eu, Hypo had a reduced pressor response (5.2 vs. 20.1%), an attenuated pulse pressure response (19.3 vs. 35.4%), and a more robust bradycardia (-17.7 vs. -7.0%) in response to DOI. These differences were atropine sensitive. In Exp III, Hypo had larger decrements in mean arterial pressure (-9.0 vs. -5.1%), heart rate ( -13.9 vs. - 7.7%), and body temperature (-4.5 vs. -2.7%) in response to 8-OH-DPAT in comparison to Eu. Parasympathetic involvement in the differential responses to 8-OH-DPAT was less clear than with DOI. Deranged autonomic control in hypothyroidism may be caused, in part, by changes in central serotonergic activity.
