Abstract The management of diabetes in a manner offering autonomous insulin therapy responsive to glucose‐directed need, and moreover with a dosing schedule amenable to facile administration, remains an ongoing goal to improve the standard of care. While basal insulins with reduced dosing frequency, even once‐weekly administration, are on the horizon, there is still no approved therapy that offers glucose‐responsive insulin function. Herein, a nanoscale complex combining both electrostatic‐ and dynamic‐covalent interactions between a synthetic dendrimer carrier and an insulin analogue modified with a high‐affinity glucose‐binding motif yields an injectable insulin depot affording both glucose‐directed and long‐lasting insulin availability. Following a single injection, it is even possible to control blood glucose for at least one week in diabetic swine subjected to daily oral glucose challenges. Measurements of serum insulin concentration in response to challenge show increases in insulin corresponding to elevated blood glucose levels, an uncommon finding even in preclinical work on glucose‐responsive insulin. Accordingly, the subcutaneous nanocomplex that results from combining electrostatic‐ and dynamic‐covalent interactions between a modified insulin and a synthetic dendrimer carrier affords a glucose‐responsive insulin depot for week‐long control following a single routine injection.
Normal metabolic health and sirtuin1 (SIRT1) by its deacetylase activity maintain voltage-gated Ca2+ channel (VGCC) function that promotes gene expression to maintain contractile coronary smooth muscle (CSM) phenotype. MetS impairs VGCC function and sarcoplasmic reticulum (SR) Ca2+ release, contributing to dedifferentiation of CSM to proliferative and osteogenic phenotypes.MetS and impaired SIRT1 will impair VGCC and SR Ca2+ release channels in CSM.Using CRISPR/Cas9 methodology a point mutation (SIRT1L100P ) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation (Ac) of Ca2+ transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. CSM cells were enzymatically dispersed and Ca2+ measured with fura-2. Depolarization with 80 mM K+ assessed Ca2+ influx through VGCC and the peak Ca2+ response to 5 mM caffeine to open SR Ca2+ release channels assessed the caffeine-sensitive SR Ca2+ store.Two-way ANOVA showed SIRT1 mutation (p=0.02) and MetS diet (p<0.0001) significantly decreased VGCC function independently, but not additively or synergistically. Interaction of SIRT1 genotype and MetS diet was significant (p<0.0001). No significant effect of genotype or diet was observed on SR calcium store release.SIRT1L100P mutation is likely to contribute to coronary atherosclerosis and SIRT1 activators may be effective therapies.
The vascular endothelium is now recognized as an important organ in the non-neural regulation of vascular tone. In particular, it is now well established that endothelial cells release various paracrine factors that relax as well as contract vascular smooth muscle cells. The relaxing factors include endothelium-derived relaxing factor (EDRF), which has been demonstrated to be nitric oxide or a nitric oxide-containing compound, prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor (EDHF). The chemical properties and nature of EDHF are still unknown. Endothelial cells also release vasoconstrictor peptides, the endothelins, which increase smooth muscle Ca 2+ concentration, resulting in potent vasoconstriction. Since the formation of all these endothelium-derived factors is due to changes in endothelial free Ca2+, the main purpose of the present review is to summarize current knowledge of the underlying molecular mechanisms involved in agonist-induced Ca2+ regulation and the mechanisms by which endothelial Ca 2+ concentrations regulate the formation of endothelium-derived vasoactive factors.
The effect of insulin to attenuate the Ca2+ and contractile response of vascular smooth muscle to a number of agonists has been described previously, but the Ca2+ regulatory mechanisms of insulin action remain unclear. We determined the effect of a physiological insulin concentration (300 pmol/l) on the Ca2+ response of vascular smooth muscle cells of the porcine right coronary artery to endothelin 1 (ET-1); furthermore, we examined the cellular Ca2+ stores affected by insulin (i.e., Ca2+ stores releasable by inositol 1,4,5-trisphosphate, caffeine, and ionomycin). We measured the Ca2+ responses of acutely isolated single smooth muscle cells with the fluorescent Ca2+ indicator Fura-2. Acute insulin exposure (20 min) significantly attenuated the Ca2+ response of single smooth muscle cells to 10 nmol/l ET-1. This inhibitory effect of insulin was observed both in the presence and absence of extracellular Ca2+. In contrast with the effects on ET-1-induced Ca2+ responses, insulin did not inhibit the Ca2+ response to 5 mmol/l caffeine, an agent that directly releases sarcoplasmic reticulum Ca2+ stores. Insulin was also without effect on the total cellular Ca2+ store released by 1 micromol/l ionomycin, a Ca2+-transporting ionophore. When ET-1 and caffeine were given in succession, a sizable caffeine-sensitive Ca2+ store could be released from insulin-treated cells but not control cells, indicating that the sarcoplasmic reticulum Ca2+ store of insulin-treated cells was not depleted by ET-1. Generalized depletion of the sarcoplasmic reticulum Ca2+ store is not one of the cellular mechanisms involved in the effect of insulin on coronary smooth muscle; instead, the effect may be due to an inhibitory influence on transmembrane signal transduction, such as diminished ET-1-induced inositol 1,4,5-trisphosphate production or reduced ability of this phosphoinositol to release stored Ca2+.
Arterial injury models for coronary artery disease have demonstrated an enhanced expression and function of either the endothelin(A) or endothelin(B) (ET(A) or ET(B)) receptor subtype. We hypothesized that organ culture would enhance the physiological function of ET receptors in the porcine right coronary artery. Arteries were either cold stored (4 degrees C) or organ cultured (37 degrees C) for 4 days. After 4 days, the artery was either 1) sectioned into rings to measure the ET-1-induced isometric tension response (3 x 10(-10)-3 x 10(-7) M), or 2) enzymatically dispersed and the isolated smooth muscle cells imaged using fura-2 to measure the myoplasmic calcium (Ca(m)) response to 3 x 10(-8) M ET-1 ( approximately EC(50)). Isometric tension and Ca(m) to ET-1 were measured in the absence and presence of bosentan (nonselective ET(A) or ET(B) receptor antagonist), BQ788 (ET(B)-selective antagonist), and BQ123 (ET(A)-selective antagonist). Compared with cold storage, organ culture induced a 2-fold increase in tension development (3 x 10(-7) M ET-1) and Ca(m) (3 x 10(-8) M ET-1), which was inhibited with bosentan, thus confirming the enhanced responses to ET-1 were due to ET receptor activation. BQ123 also inhibited the enhanced contraction and Ca(m) responses to ET-1. In contrast, BQ788 failed to inhibit tension development and Ca(m) responses to ET-1 in organ culture and cold storage. Sarafotoxin 6C (ET(B) agonist) failed to elicit an increased Ca(m) response in organ culture compared with cold storage. Our results indicate the increased tension development and Ca(m) responses to ET-1 in organ culture are attributable to ET(A) receptors, and not ET(B) receptors.
1. We tested the hypothesis that the sarcoplasmic reticulum (SR) buffers (attenuates) the increase in averaged myoplasmic free [Ca2+] (Ca(im)) resulting from Ca2+ influx. 2. Fura-2 measurements of Ca(im) were obtained in single smooth muscle cells freshly dispersed from bovine coronary artery. 3. Caffeine (5 x 10(-3) M) elicited a transient increase in Ca(im) and depleted the SR Ca2+ store. In the continued presence of caffeine or 10(-5) M-ryanodine SR buffering of Ca(im) was inhibited. Subsequent exposure to high extracellular [K+] (greater than 30 mM, equimolar Na+ removal) elicited a 2-fold more rapid and 2-fold greater peak increase in Ca(im) than high K+ elicited when SR buffering of Ca(im) was normal. The augmented increase in Ca(im) was inhibited 35% by 10(-5) M-diltiazem, 65% by 2 x 10(-4) M-LaCl3, and 87% in Ca(2+)-free external solution. 4. When Ca(im) buffering capacity was increased by partially depleting the SR with a transient (1 min) exposure to caffeine, subsequent exposure to 80 nM-K+ solution increased Ca(im) almost 2-fold more slowly than 80 mM-K+ before depletion of Ca2+ from the SR. However, the influxing Ca2+ was sequestered by the SR and refilled it, as evident by the subsequent caffeine-induced Ca(im) transient being identical to the first. Increasing extracellular [K+] (thus, increasing depolarization and Na+ removal) caused proportional increases in Ca(im) and the subsequent caffeine-induced Ca(im) transients were proportionally larger, indicating a graded filling of the SR by Ca2+ influx. 5. Diltiazem (10(-5) M) inhibited the refilling of the SR achieved by 80 mM-K+, by 26%. Refilling was inhibited 76% by 80 mM-K+, Ca(2+)-free solution, indicating the fraction of refilling dependent on influx of Ca2+ through voltage-gated Ca2+ channels, leak channels, and other influx pathways. Mild depolarization with 35 mM-K+ (no Na+ removal) often caused no increase in Ca(im), but influx through voltage-gated Ca2+ channels occurred because the SR Ca2+ store was refilled. Also, 10(-5) M-diltiazem or 10(-6) M-TA3090 inhibited the refilling to levels attributable only to leak influx of Ca2+. 6. All data support our hypothesis that the SR significantly attenuates the amount of Ca2+ influx that accumulates to increase Ca(im).(ABSTRACT TRUNCATED AT 400 WORDS)
