Nitric Oxide Mediates Stretch-Induced Ca2+ Release via Activation of Phosphatidylinositol 3-Kinase-Akt Pathway in Smooth Muscle

Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca(2+) release occurs in the form of Ca(2+) sparks and Ca(2+) waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown.We examined the transduction mechanism linking cell stretch to Ca(2+) release. The probability and frequency of Ca(2+) sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca(2+) sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204+/-28.6% and 258+/-36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level.Taking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle

PI(3)k-Akt mediates stretch- induced Ca2+ release in smooth myocytes.

<p>Linescan images show the effect of LY492002 on Ca2+ spark at stretch length ΔL = 18%. LY492002 could not completely abrogated the immediately stretch-induced Ca2+ sparks (A), but completely abolished Ca2+ sparks occurred in stretch-maintaining stage (B). C, representatives of experiments in the absence and the presence of another PI3 kinase inhibitor, wortmannin. Like LY492002, wortmannin entirely abrogated Ca2+ sparks occurred in the stretch-maintaining stage. C, summary data of Ca2+ spark probability after sequential stretch. The numbers indicate the response experiments to stretch out of all experiments.</p

Stretch-induced NO production in single cell and intact smooth muscle tissues.

<p>A, ESR spectra of NO trapped by DETC-iron (II) complex in mouse bladder smooth muscle strips. Compared to control (slack tissue strips, gray line), NO was greatly increased in stretched tissue strips (black line). The inset shows the magnetic field range of NO in the presence of SNP. B, summary data of NO production. Note the significantly difference of NO production between the control group and stretch group; * P<0.01, n = 6. C, DAF-2 single cell experiments: by stretch the cell length to ΔL = 9% DAF-2 fluorescence transient occurred immediately. D, upper is x-y images taken from a tissue strip incubated with DAF-2; the lower is pseudo linescan images taken from a series of x-y image obtained from before (left) and after (right) stretch of the intact mouse bladder smooth muscle strip incubated with DAF-2, and at below of pseudo linescan images show NO transient profiles taken from slack and stretch. E, before ( left ) and after ( right ) stretch of tissue segment in the presence of L-name. Note that the stretch-induced NO production was completely inhibited by L-name.</p

Ca2+ spark property was not altered by ODQ.

<p>A, sample of linescan images obtained from stretched smooth myocytes. Compared to control (A, left), there was no significant change of the Ca2+ spark probability (A, right), frequency, and peak Ca2+ in the presence of ODQ. B, summary data of probability (a, n = number of experiments), frequency (b, n = number of experiments), and peak Ca2+ (c, n = number of sparks) of Ca2+ sparks. C & D, profiles and summary data show the relationship of NO and stretch-induced Ca2+ spark, indicating NO always occurred prior to Ca2+ sparks (n = number of experiments, *P<0.05).</p

eNOS gene deletion alters the property of stretch- induced Ca2+ sparks in smooth myocytes.

<p>A, representative x-y images recorded from a wildtype (upper) and an eNOS deficient smooth myocyte (lower). The stretch-dependence of Ca2+-spark frequency is present in fluo-4-loaded smooth myocytes from the wild type (WT, 15 out of 18 experiments) but absent in those from eNOS-deficient mice (0 out of 21 experiments); the below of the images are profiles of Ca2+ ratio from WT (black) and eNOS null (gray) cells. B, sample of x-y images recorded from membrane voltage depolarized WT (upper) and eNOS knockout (lower) smooth myocytes; compared to WT cells, the eNOS deletion cells have a low Ca2+ spark probability (see also D, right). C, linescan images recorded from WT (left), eNOS null cells (middle) as well as in the presence of SNAP (right). D, summary data of Ca2+ spark probability in WT and eNOS deficient smooth myocytes.</p

Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains

Sinoatrial node (SAN) cells are the heart's primary pacemaker. Their activity is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR pathway that catalyzes the production of cAMP. There are current gaps in our knowledge regarding the dominant AC isoforms and the specific roles of Ca2+-activated ACs in the SAN. The current study tests the hypothesis that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within microdomains to orchestrate heart rate regulation during β-AR signaling. In contrast to atrial and ventricular myocytes, SAN cells express a diverse repertoire of ACs, with ACI as the predominant Ca2+-activated isoform. Although ACI-KO (ACI-/-) mice exhibit normal cardiac systolic or diastolic function, they experience SAN dysfunction. Similarly, SAN-specific CRISPR/Cas9-mediated gene silencing of ACI results in sinus node dysfunction. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels form functional microdomains almost exclusively with ACI, while ryanodine receptor and L-type Ca2+ channels likely compartmentalize with ACI and other AC isoforms. In contrast, there were no significant differences in T-type Ca2+ and Na+ currents at baseline or after β-AR stimulation between WT and ACI-/- SAN cells. Due to its central characteristic feature as a Ca2+-activated isoform, ACI plays a unique role in sustaining the rise of local cAMP and heart rates during β-AR stimulation. The findings provide insights into the critical roles of the Ca2+-activated isoform of AC in sustaining SAN automaticity that is distinct from contractile cardiomyocytes

Genetic dissection of ion currents underlying all-or-none action potentials in C. elegans body-wall muscle cells

Although the neuromuscular system of C. elegans has been studied intensively, little is known about the properties of muscle action potentials (APs). By combining mutant analyses with in vivo electrophysiological recording techniques and Ca2+ imaging, we have established the fundamental properties and molecular determinants of body-wall muscle APs. We show that, unlike mammalian skeletal muscle APs, C. elegans muscle APs occur in spontaneous trains, do not require the function of postsynaptic receptors, and are all-or-none overshooting events, rather than graded potentials as has been previously reported. Furthermore, we show that muscle APs depend on Ca2+ entry through the L-type Ca2+ channel EGL-19 with a contribution from the T-type Ca2+ channel CCA-1. Both the Shaker K+ channel SHK-1 and the Ca2+/Cl−-gated K+ channel SLO-2 play important roles in controlling the speed of membrane repolarization, the amplitude of afterhyperpolarization (AHP) and the pattern of AP firing; SLO-2 is also important in setting the resting membrane potential. Finally, AP-elicited elevations of [Ca2+]i require both EGL-19 and the ryanodine receptor UNC-68. Thus, like mammalian skeletal muscle, C. elegans body-wall myocytes generate all-or-none APs, which evoke Ca2+ release from the sarcoplasmic reticulum (SR), although the specific ion channels used for AP upstroke and repolarization differ