Subject(s)
Acupuncture Therapy , Health Knowledge, Attitudes, Practice , Patient Acceptance of Health Care , Acupuncture , Acupuncture Therapy/statistics & numerical data , Adolescent , Adult , Female , General Practice , Health Care Surveys , Health Services Needs and Demand , Humans , Male , State Medicine , Surveys and Questionnaires , United Kingdom , Young AdultABSTRACT
It is now established that non-contractile cells with thin filopodia, also called vascular interstitial cells (VICs), are constitutively present in the media of many, if not all, blood vessels. The aim of this study was to determine the type of cell lineage to which arterial VICs belong using immunocytochemical, and real-time and reverse transcription PCR (RT-PCR). Using RT-PCR, we compared gene expression profiles of single VICs and smooth muscle cells (SMCs) freshly dispersed from rat middle cerebral artery. Both VICs and SMCs expressed the SMC marker, smooth muscle myosin heavy chain (SM-MHC), but did not express fibroblast, pericyte, neuronal, mast cell, endothelial or stem cell markers. Freshly isolated VICs also did not express c-kit, which is the marker for interstitial cells of Cajal in the gastrointestinal tract. Immunocytochemical labelling of contractile proteins showed that VICs and SMCs expressed SM-MHC similarly to the same degree, but VICs in contrast to SMCs had decreased expression of alpha-SM-actin and very low or no expression of calponin. Real-time RT-PCR was consistent with immunocytochemical experiments and showed that VICs had four times lower gene expression of calponin comparing to SMCs, which may explain VICs' inability to contract. VICs had greater expression than SMCs of structural proteins such as non-muscular beta-actin and desmin. The results obtained suggest that VICs represent a subtype of SMCs and may originate from the same precursor as SMCs, but later develop filopodia and a non-contractile cell phenotype.
Subject(s)
Middle Cerebral Artery/cytology , Myocytes, Smooth Muscle/cytology , Animals , Contractile Proteins/genetics , Contractile Proteins/metabolism , Gene Expression Regulation , Immunohistochemistry , Male , Middle Cerebral Artery/ultrastructure , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/ultrastructure , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A voltage-gated Na+ current was characterised in freshly dissociated mouse portal vein (PV) smooth muscle myocytes. The current was found superimposed upon the relatively slow L-type Ca2+ current and was resistant to conventional Ca2+ channel blockers but was abolished by external Na+ replacement and tetrodotoxin (TTX, 1 microM). The molecular identity of the channel responsible for this conductance was determined by RT-PCR where only the transcripts for Na+ channel genes SCN7a, 8a and 9a were detected. The presence of the protein counterparts to the SCN8a and 9a genes (NaV1.6 and NaV1.7, respectively) on the individual smooth muscle myocytes were confirmed in immunocytochemistry, which showed diffuse staining around a predominantly plasmalemmal location. TTX inhibited the action potential in individual myocytes generated in the current clamp mode but isometric tissue tension experiments revealed that TTX (1 and 5 microM) had no effect on the inherent mouse PV rhythmicity. However, the Na+ channel opener veratridine (10 and 50 microM) significantly increased the length of contraction and the interval between contractions. This effect was not influenced by pre-incubation with atropine, prazosin and propranolol, but was reversed by TTX (1 microM) and completely abolished by nicardipine (1 microM). Furthermore, preincubation with the reverse-mode Na+-Ca2+ exchange blocker KB-R7943 (10 microM) also inhibited the veratridine response. We have established for the first time the molecular identity of the voltage-gated Na+ channel in freshly dispersed smooth muscle cells and have shown that these channels can modulate contractility through a novel mechanism of action possibly involving reverse mode Na+-Ca2+ exchange.
