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Background and purpose In cardiac myocytes, cyclic AMP (cAMP) produced by both β1 - and β2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by β1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. Experimental approach The cAMP responses produced by β1 -and β2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex. Key results Selective activation of β1 - or β2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of β1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of β2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4. Conclusion and implications These results support the conclusion that cAMP produced by β2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

Activation of different receptors that act by generating the common second messenger cyclic adenosine monophosphate (cAMP) can elicit distinct functional responses in cardiac myocytes. Selectively sequestering cAMP activity to discrete intracellular microdomains is considered essential for generating receptor-specific responses. The processes that control this aspect of compartmentalized cAMP signaling, however, are not completely clear. Over the years, technological innovations have provided critical breakthroughs in advancing our understanding of the mechanisms underlying cAMP compartmentation. Some of the factors identified include localized production of cAMP by differential distribution of receptors, localized breakdown of this second messenger by targeted distribution of phosphodiesterase enzymes, and limited diffusion of cAMP by protein kinase A (PKA)-dependent buffering or physically restricted barriers. The aim of this review is to provide a discussion of our current knowledge and highlight some of the gaps that still exist in the field of cAMP compartmentation in cardiac myocytes.

In article number 1903140, Surya M. Nauli and co‐workers discover ciliary extracellular‐like vesicles (cELVs). Released by fluid‐shear, cELVs act as nano‐compartments within a cilium. Proteomic identification reveals the role of cELVs in cardiovascular functions. [Image: see text]

Primary cilia are shown to have membrane swelling, also known as ciliary bulbs. However, the role of these structures and their physiological relevance remains unknown. Here, it is reported that a ciliary bulb has extracellular vesicle (EV)‐like characteristics. The ciliary extracellular‐like vesicle (cELV) has a unique dynamic movement and can be released by mechanical fluid force. To better identify the cELV, differential multidimensional proteomic analyses are performed on the cELV. A database of 172 cELV proteins is generated, and all that examined are confirmed to be in the cELV. Repressing the expression of these proteins in vitro and in vivo inhibits cELV formation. In addition to the randomized heart looping, hydrocephalus, and cystic kidney in fish, compensated heart contractility is observed in both fish and mouse models. Specifically, low circulation of cELV results in hypotension with compensated heart function, left ventricular hypertrophy, cardiac fibrosis, and arrhythmogenic characteristics, which result in a high mortality rate in mice. Furthermore, the overall ejection fraction, stroke volume, and cardiac output are significantly decreased in mice lacking cELV. It is thus proposed that the cELV as a nanocompartment within a primary cilium plays an important role in cardiovascular functions., The ciliary extracellular‐like vesicle (cELV) acts as a nanocompartment within a cilium. The cELV increases the flexural rigidity of a cilium and can be released by mechanical fluid‐shear force. Proteomic identification of the cELV reveals its physiological roles in zebrafish and mouse models. In addition to the extracardiac phenotypes, the cELV is required for cardiovascular functions.

Patients with polycystic kidney disease (PKD) are characterized with uncontrolled hypertension. Hypertension in PKD is a ciliopathy, an abnormal function and/or structure of primary cilia. Primary cilia are cellular organelles with chemo and mechanosensory roles. In the present studies, we designed a cilia-targeted (CT) delivery system to deliver fenoldopam specifically to the primary cilia. We devised the iron oxide nanoparticle (NP)-based technology for ciliotherapy. Live imaging confirmed that the CT-Fe(2)O(3)-NPs specifically targeted primary cilia in cultured cells in vitro and vascular endothelia in vivo. Importantly, the CT-Fe(2)O(3)-NPs enabled the remote control of the movement and function of a cilium with an external magnetic field, making the nonmotile cilium exhibit passive movement. The ciliopathic hearts displayed hypertrophy with compromised functions in left ventricle pressure, stroke volume, ejection fraction, and overall cardiac output because of prolonged hypertension. The CT-Fe(2)O(3)-NPs significantly improved cardiac function in the ciliopathic hypertensive models, in which the hearts also exhibited arrhythmia, which was corrected with the CT-Fe(2)O(3)-NPs. Intraciliary and cytosolic Ca(2+) were increased when cilia were induced with fluid flow or magnetic field, and this served as a cilia-dependent mechanism of the CT-Fe(2)O(3)-NPs. Fenoldopam-alone caused an immediate decrease in blood pressure, followed by reflex tachycardia. Pharmacological delivery profiles confirmed that the CT-Fe(2)O(3)-NPs were a superior delivery system for targeting cilia more specifically, efficiently, and effectively than fenoldopam-alone. The CT-Fe(2)O(3)-NPs altered the mechanical properties of nonmotile cilia, and these nano-biomaterials had enormous clinical potential for ciliotherapy. Our studies further indicated that ciliotherapy provides a possibility toward personalized medicine in ciliopathy patients.

BACKGROUND AND PURPOSE: In cardiac myocytes, cAMP produced by both β(1) and β(2)-adrenergic receptors (ARs) results in an increase in L-type Ca(2+) channel activity and myocyte contraction. However, only cAMP produced by β(1)ARs is able to enhance myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2). The purpose of this study was to test the hypothesis that β(2)AR stimulation produces a cAMP signal that is unable to reach SERCA2, and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. EXPERIMENTAL APPROACH: The cAMP responses produced by β(1) and β(2)AR stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors: Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell, and Epac2-αKAP, which is targeted to the SERCA2 signaling complex. KEY RESULTS: Selective activation of β(1) or β(2)ARs produces cAMP responses detected by Epac2-camps. However, only β(1)AR stimulation produces a cAMP response detected by Epac2-αKAP. Yet, β(2)AR stimulation was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibition of PDE2 or PDE3, but not PDE4. CONCLUSION AND IMPLICATIONS: These results support the conclusion that cAMP produced by β(2)AR stimulation is not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. Leveraging this discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

Ciliopathies caused by abnormal function of primary cilia include expanding spectrum of kidney, liver, and cardiovascular disorders. There is currently no treatment available for patients with cilia dysfunction. Therefore, we generated and compared two different (metal and polymer) cilia-targeted nanoparticle drug delivery systems (CTNDDS), CT-DAu-NPs and CT-PLGA-NPs, for the first time. These CTNDDS loaded with fenoldopam were further compared to fenoldopam-alone. Live-imaging of single-cell-single-cilium analysis confirmed that CTNDDS specifically targeted to primary cilia. While CTNDDS did not show any advantages over fenoldopam-alone in cultured cells in vitro, CTNDDS delivered fenoldopam more superior than fenoldopam-alone by eliminating the side effect of reflex tachycardia in murine models. Although slow infusion was required for fenoldopam-alone in mice, bolus injection was possible for CTNDDS. Though there were no significant therapeutic differences between CT-DAu-NPs and CT-PLGA-NPs, CT-PLGA-NPs tended to correct ciliopathy parameters closer to normal physiological levels, indicating CT-PLGA-NPs were better cargos than CT-DAu-NPs. Both CTNDDS showed no systemic adverse effect. In summary, our studies provided scientific evidence that existing pharmacological agent could be personalized with advanced nanomaterials to treat ciliopathy by targeting cilia without the need of generating new drugs.

Primary cilia are hair-like cellular extensions that sense microenvironmental signals surrounding cells. The role of adenylyl cyclases in ciliary function has been of interest because the product of adenylyl cyclase activity, cAMP, is relevant to cilia-related diseases. In the present study, we show that vasopressin receptor type-2 (V2R) is localized to cilia in kidney epithelial cells. Pharmacologic inhibition of V2R with tolvaptan increases ciliary length and mechanosensory function. Genetic knockdown of V2R, however, does not have any effect on ciliary length, although the effect of tolvaptan on ciliary length is dampened. Our study reveals that tolvaptan may have a cilia-specific effect independent of V2R or verapamil-sensitive calcium channels. Live-imaging of single cilia shows that V2R activation increases cilioplasmic and cytoplasmic cAMP levels, whereas tolvaptan mediates cAMP changes only in a cilia-specific manner. Furthermore, fluid-shear stress decreases cilioplasmic, but not cytoplasmic cAMP levels. Our data indicate that cilioplasmic and cytoplasmic cAMP levels are differentially modulated. We propose that the cilium is a critical sensor acting as a responsive cAMP microcompartment during physiologically relevant stimuli.

Small-conductance Ca(2+)-activated K(+) (SK) channels are voltage-independent and are activated by Ca(2+) binding to the calmodulin constitutively associated with the channels. Both the pore-forming subunits and the associated calmodulin are subject to phosphorylation. Here, we investigated the modulation of different SK channel subtypes by phosphorylation, using the cultured endothelial cells as a tool. We report that casein kinase 2 (CK2) negatively modulates the apparent Ca(2+) sensitivity of SK1 and IK channel subtypes by more than 5-fold, whereas the apparent Ca(2+) sensitivity of the SK3 and SK2 subtypes is only reduced by ~2-fold, when heterologously expressed on the plasma membrane of cultured endothelial cells. The SK2 channel subtype exhibits limited cell surface expression in these cells, partly as a result of the phosphorylation of its C-terminus by cyclic AMP-dependent protein kinase (PKA). SK2 channels expressed on the ER and mitochondria membranes may protect against cell death. This work reveals the subtype-specific modulation of the apparent Ca(2+) sensitivity and subcellular localization of SK channels by phosphorylation in cultured endothelial cells.

Cellular signaling represents an evolution of biological systems to sense external stimuli and communicate extracellular microenvironment to the intracellular compartments. The processes underlying molecular signaling have been widely studied due to their important cellular functions. There are numerous techniques available to quantitate the different molecules involved in cellular processes. Among them, calcium is a ubiquitous signaling molecule involved in many biological pathways. Over time the methods to measure intracellular calcium have advanced to better understand its role as a second messenger. In this chapter, we introduce a method to study a single cilium, a mechanosensor that elicits a calcium signaling cascade. To successfully observe the calcium changes in this thin cylindrical-like projection from the cell surface, we utilize a genetically encoded sensor with a high spatial and temporal resolution. In addition, the probe must be localized to the ciliary compartment in order to observe the intraciliary calcium signaling dynamics. To this end, a cilium targeting genetically encoded indicator is used to observe calcium fluxes in both cytoplasm and cilioplasm.

Primary cilia arebiophysically-sensitive organelles responsible for sensing fluid-flow and transducing this stimulus into intracellular responses. Previous studies have shown that the primary cilia mediate flow-induced calcium influx, and sensitivity of cilia function to flow is correlated to cilia length. Cells with abnormal cilia length or function can lead to a host of diseases that are collectively termed as ciliopathies. Rapamycin, a potent inhibitor of mTOR (mammalian target of rapamycin), has been demonstrated to be a potential pharmacological agent against the aberrant mTOR signaling seen in ciliopathies such as polycystic kidney disease (PKD) and tuberous sclerosis complex (TSC). Here we look at the effects of rapamycin on ciliary length and function for the first time. Compared to controls, primary cilia in rapamycin-treated porcine renal epithelial and mouse vascular endothelial cells showed a significant increase in length. Graded increases in fluid-shear stress further indicates that rapamycin enhances cilia sensitivity to fluid flow. Treatment with rapamycin led to G0 arrest in porcine epithelial cells while no significant change in cell cycle were observed in rapamycin-treated mouse epithelial or endothelial cells, indicating a species-specific effect of rapamycin. Given the previousin vitro and in vivo studies establishing rapamycin as a potential therapeutic agent for ciliopathies, such as PKD and TSC, our studies show that rapamycin enhances ciliary function and sensitivity to fluid flow. The results of our studies suggest a potential ciliotherapeutic effect of rapamycin.

Primary cilia are hair-like cellular extensions that sense microenvironmental signals surrounding cells. The role of adenylyl cyclases in ciliary function has been of interest because the product of adenylyl cyclase activity, cAMP, is relevant to cilia-related diseases. In the present study, we show that vasopressin receptor type-2 (V2R) is localized to cilia in kidney epithelial cells. Pharmacologic inhibition of V2R with tolvaptan increases ciliary length and mechanosensory function. Genetic knockdown of V2R, however, does not have any effect on ciliary length, although the effect of tolvaptan on ciliary length is dampened. Our study reveals that tolvaptan may have a cilia-specific effect independent of V2R or verapamil-sensitive calcium channels. Live-imaging of single cilia shows that V2R activation increases cilioplasmic and cytoplasmic cAMP levels, whereas tolvaptan mediates cAMP changes only in a cilia-specific manner. Furthermore, fluid-shear stress decreases cilioplasmic, but not cytoplasmic cAMP levels. Our data indicate that cilioplasmic and cytoplasmic cAMP levels are differentially modulated. We propose that the cilium is a critical sensor acting as a responsive cAMP microcompartment during physiologically relevant stimuli.

We have previously evaluated and reported numerous classes of linear and cyclic peptides containing hydrophobic and hydrophilic segments for intracellular delivery of multiple molecular cargos. Herein, a combination of histidine and tryptophan amino acids were designed and evaluated for their efficiency in intracellular delivery of cell-impermeable phosphopeptides and the anti-HIV drug, emtricitabine. Two new decapeptides, with linear and cyclic natures, both containing alternate tryptophan and histidine residues, were synthesized using Fmoc/tBu solid-phase chemistry. The peptides were characterized and purified by using matrix-assisted laser desorption/ionization (MALDI) spectroscopy and high-performance liquid chromatography (HPLC), respectively. These peptides did not show significant toxicity up to 100 &micro, M in ovarian cancer (SK-OV-3) and leukemia cancer (CCRF-CEM) cells. Furthermore, the cellular uptake of a fluorescence (F&rsquo, )-labeled cell-impermeable phosphopeptide (F&rsquo, GpYEEI) was enhanced in the presence of linear (WH)5 and cyclic [WH]5 by 2- and 8-fold, respectively, compared to the uptake of the phosphopeptide alone. The cellular uptake was not significantly changed in the presence of endocytosis inhibitors. Furthermore, the intracellular uptake of the fluorescently-labeled anti-HIV drug, emtricitabine (F&rsquo, FTC), by linear (WH)5 and cyclic [WH]5 in SK-OV-3 cancer cell lines was found to be enhanced by 3.5- and 9-fold, respectively, compared to that of the drug alone. Fluorescent uptake experiments confirmed the localization of F&rsquo, GpYEEI-loaded cyclic [WH]5 intracellularly in the SK-OV-3 cancer cell line after 3 h of incubation. Thus, these data demonstrated that [WH]5 containing tryptophan and histidine enhanced the cellular uptake of F&rsquo, GpYEEI and emtricitabine.