Abstract
Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.
Highlights
Encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function
We report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle
We report the production of transgenic mice expressing G-CaMP under control of the smooth muscle myosin heavy chain promoter and the use of these mice to resolve nerve-evoked, postsynaptic signaling in smooth muscle
Summary
19, 137–141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2؉ reporter Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2؉ signals, mediated by distinct neurotransmitters. Encoded cell reporters of cell signaling hold great promise for the study of in vivo cell physiology. This field has advanced rapidly recently with the development of fluorescence resonance energy transfer-based probes such as cameleons [2], fluorescent proteins that can be modified following expression [3], and re-engineered fluorescent proteins [1, 4] (for review, see Ref. 5). G-CaMP-signaling mice provide an effective tool to examine Ca2ϩ signaling in single lineages from complex tissues
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