The TSC22D, WNK, and NRBP gene families exhibit functional buffering and evolved with Metazoa for cell volume regulation.

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.

Shellfish CO2 excretion is modulated by seawater carbonate chemistry but largely independent of pCO2.

Four species of shellfish, blue mussel (Mytilus galloprovincialis), Pacific abalone (Haliotis discus hannai), zhikong scallops (Chlamys farreri), and Pacific oyster (Crassostrea gigas), were exposed to decoupled carbonate system variables to investigate the impacts of different seawater carbonate parameters on the CO2 excretion process of mariculture shellfish. Six experimental groups with two levels of seawater pH (pH 8.1 and pH 7.7) and three levels of total alkalinity (TA = 1000, 2300, and 3600 µmol/kg, respectively) were established, while pH 8.1 and TA = 2300 µmol/kg was taken as control. Results showed that the CO2 excretion rates of these tested shellfish were significantly affected by the change in carbonate chemistry (P < 0.05). At the same TA level, animals incubated in the acidified group (pH 7.7) had a lower CO2 excretion rate than those in the control group (pH 8.1). In comparison, at the same pH level, the CO2 excretion rate increased when seawater TA level was elevated. No significant correlation between the CO2 excretion rate and seawater pCO2 levels (P > 0.05) was found; however, a significant correlation (P < 0.05) between CO2 excretion rate and TA-DIC (the difference between total alkalinity and dissolved inorganic carbon) was observed. Blue mussel has a significantly higher CO2 excretion rate than the other three species in the CO2 excretions per unit mass of soft parts, with no significant difference observed among these three species. However, in terms of CO2 excretion rate per unit mass of gills, abalone has the highest CO2 excretion rate, while significant differences were found between each species. Our studies indicate that the CO2 buffering capacity impacts the CO2 excretion rate of four shellfish species largely independent of pCO2. Since CO2 excretion is related to acid-base balancing, the results imply that the effects of other carbonate parameters, particularly the CO2 buffering capacity, should be studied to fully understand the mechanism of how acidification affects shellfish. Besides, the species difference in gill to soft parts proportion may contribute to the species difference in responding to ocean acidification.

Activity-dependent extracellular potassium changes in unmyelinated versus myelinated areas in olfactory regions of the isolated female guinea-pig brain.

The potassium released in the extracellular space during neuronal activity is rapidly removed by glia and neurons to maintain tissue homeostasis. Oligodendrocyte-derived myelin axonal coating contributes to potassium buffering and is therefore crucial to control brain excitability. We studied activity-dependent extracellular potassium ([K+]o) changes in the piriform cortex (PC), a region that features highly segregated bundles of myelinated and unmyelinated fibers. Four-aminopyridine (4AP; 50 µM) treatment or patterned high-frequency stimulations (hfST) were utilized to generate [K+]o changes measured with potassium-sensitive electrodes in the myelinated lateral olfactory tract (LOT), in the unmyelinated PC layer I and in the myelinated deep PC layers in the ex vivo isolated guinea-pig brain. Seizure-like events induced by 4AP are initiated by the abrupt [K+]o rise in the layer I formed by unmyelinated fibers (Uva et al., 2017). Larger [K+]o shifts occurred in unmyelinated layers compared to the myelinated LOT. LOT hfST that mimicks pre-seizure discharges also generated higher [K+]o changes in unmyelinated PC layer I than in LOT and deep PC layers. The treatment with the Kir4.1 potassium channel blocker BaCl2 (100 µM) enhanced the [K+]o changes generated by hfST in myelinated structures. Our data show that activity-dependent [K+]o changes are intrinsically different in myelinated vs unmyelinated cortical regions. The larger [K+]o shifts generated in unmyelinated structures may represent a vehicle for seizure generation.

ß-Alanine Plus Sodium Bicarbonate Co-Supplementation Does Not Decrease Neuromuscular Fatigue in Swimming.

This study aimed to investigate the effects of chronic ß-alanine (ßA) plus acute sodium bicarbonate (SB) co-supplementation on neuromuscular fatigue during high-intensity intermittent efforts in swimming. Eleven regional and national competitive-level young swimmers performed a neuromuscular fatigue assessment before and immediately after two 20 × 25-m front crawl maximal efforts every 90 s, performed at pre- and post-4-week co-supplementation. Neuromuscular fatigue was evaluated by percutaneous electrical stimuli through the twitch interpolation technique on the triceps brachii and quadriceps femoris. Performance was defined by the mean time of the 20 efforts and blood samples to lactate concentrations were collected every four efforts. Participants supplemented 3.2-6.4 g·day-1 of chronic ßA or placebo (PL) during four weeks, and acute 0.3 g·kg-1 of SB or PL 60 min before the second assessment (allowing ßA+SB and PL+PL groups). No statistical changes were found in neuromuscular fatigue of triceps brachii. In the quadriceps femoris, a main effect of time was found in potentiated twitch delta values in pooled groups, showing a statistical increase of 19.01% after four weeks (Δ = 13.05 [0.35-25.75] N; p = 0.044), without time × group interactions. No statistical difference was found in the swimming performance. Blood lactate increased by 25.06% only in the ßA+SB group (Δ = 6.40 [4.62-8.18] mM; p Bonf < 0.001) after the supplementation period. In conclusion, 4-week ßA and SB co-supplementation were not able to reduce neuromuscular fatigue levels and improve performance in highintensity intermittent efforts, but statistically increased blood lactate levels.

Psychological distress and alcohol use disorder in college students of color: Social support's moderating properties.

Alcohol use is prevalent among college students. Research has found that psychological distress in the form of depression, anxiety, or loneliness has been consistently associated with greater alcohol use. Because Students of Color (SoC) disproportionately experience greater psychological distress than White students, it is critical to determine buffers against psychological distress and subsequent alcohol use consequences. Previous literature found that social support can protect against the effects of psychological distress and weaken its link with alcohol use. This study aimed to determine the moderating effect of social support in the relation between psychological distress and alcohol use among SoC. College Students of Color from across the U.S. (n = 211, Mage = 27.51, SD = 9.63) were recruited via Prolific to answer an online survey through Qualtrics. Linear regression analyses showed that psychological distress variables (i.e., depression, anxiety, and loneliness) were positively associated with alcohol use. However, moderation analysis did not find any form of social support to moderate the relation between psychological distress variables and alcohol use. Future research needs to identify other protective factors against alcohol use to support SoC in their academic journey.

Freezing-induced acidification of sea ice brine.

The acidity of sea ice and snow plays a key role in the chemistry of the cryosphere; an important example lies in the photochemical catalytic release of reactive bromine in polar regions, facilitated at pHs below 6.5. We apply in-situ acid-base indicators to probe the microscopic acidity of the brine within the ice matrix in artificial sea water at a range of concentrations (0.35-70 PPT) and initial pHs (6-9). The results are supported by analogous measurements of the most abundant salts in seawater: NaCl, Na2SO4, and CaCO3. In the research herein, the acidity is expressed in terms of the Hammett acidity function, H2-. The obtained results show a pronounced acidity increase in sea water after freezing at -15 °C and during the subsequent cooling down to -50 °C. Importantly, we did not observe any significant hysteresis; the values of acidity upon warming markedly resembled those at the corresponding temperatures at cooling. The acidity increase is attributed to the minerals' crystallization, which is accompanied by a loss of the buffering capacity. Our observations show that lower salinity sea water samples (≤ 3.5 PPT) reach pH values below 6.5 at the temperature of -15 °C, whereas higher salinity ices attain such values only at -30 °C. The ensuing implications for polar chemistry and the relevance to the field measurements are discussed.

Cell-Impermeable Buffering Fluorogenic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Morphology Dynamics.

Super-resolution fluorescence imaging has emerged as a potent tool for investigating the nanoscale structure and function of the plasma membrane (PM). Nevertheless, the challenge persists in achieving super-resolution imaging of PM dynamics due to limitations in probe photostability and issues with cell internalization staining. Herein, we report assembly-mediated buffering fluorogenic probes BMP-14 and BMP-16 exhibiting fast PM labeling and extended retention time (over 2 h) on PM. The incorporation of alkyl chains proves effective in promoting the aggregation of BMP-14 and BMP-16 into nonfluorescent nanoparticles to realize fluorogenicity and regulate the buffering capacity to rapidly replace photobleached probes ensuring stable long-term super-resolution imaging of PM. Utilizing these PM-buffering probes, we observed dynamic movements of PM filopodia and continuous shrinkage, leading to the formation of extracellular vesicles (EVs) using structured illumination microscopy (SIM). Furthermore, we discovered two distinct modes of EV fusion: one involving fusion through adjacent lipids and the other through filamentous lipid traction. The entire process of EV fusion outside the PM was dynamically tracked. Additionally, BMP-16 exhibited a unique capability of inducing single-molecule fluorescence blinking when used for cell membrane staining. This property makes BMP-16 suitable for the PAINT imaging of cell membranes.

Mitoferrin2 is a synthetic lethal target for chromosome 8p deleted cancers.

BACKGROUND: Somatic copy number alterations are a hallmark of cancer that offer unique opportunities for therapeutic exploitation. Here, we focused on the identification of specific vulnerabilities for tumors harboring chromosome 8p deletions. METHODS: We developed and applied an integrative analysis of The Cancer Genome Atlas (TCGA), the Cancer Dependency Map (DepMap), and the Cancer Cell Line Encyclopedia to identify chromosome 8p-specific vulnerabilities. We employ orthogonal gene targeting strategies, both in vitro and in vivo, including short hairpin RNA-mediated gene knockdown and CRISPR/Cas9-mediated gene knockout to validate vulnerabilities. RESULTS: We identified SLC25A28 (also known as MFRN2), as a specific vulnerability for tumors harboring chromosome 8p deletions. We demonstrate that vulnerability towards MFRN2 loss is dictated by the expression of its paralog, SLC25A37 (also known as MFRN1), which resides on chromosome 8p. In line with their function as mitochondrial iron transporters, MFRN1/2 paralog protein deficiency profoundly impaired mitochondrial respiration, induced global depletion of iron-sulfur cluster proteins, and resulted in DNA-damage and cell death. MFRN2 depletion in MFRN1-deficient tumors led to impaired growth and even tumor eradication in preclinical mouse xenograft experiments, highlighting its therapeutic potential. CONCLUSIONS: Our data reveal MFRN2 as a therapeutic target of chromosome 8p deleted cancers and nominate MFNR1 as the complimentary biomarker for MFRN2-directed therapies.

Heart and shoal: Social cues and oxytocin receptors impact stress recovery in the zebrafish.

In many species, social interactions decrease behavioral, hormonal, and neural responses to environmental stressors. While "social buffering" and its mechanisms have received considerable attention in mammals, we know less about the phenomenon in fish. The nonapeptide oxytocin regulates social behavior across vertebrates and plays an important role in social buffering in mammals. We investigated social buffering in the zebrafish by evaluating how the social environment and oxytocin receptors impact recovery from an acute stressor. Male and female fish were briefly exposed to alarm substance and recovered either in isolation or within view of a stimulus shoal. Alarm substance did not increase social approach, but social stimuli improved behavioral stress recovery. Oxytocin receptor antagonism decreased social approach during stress recovery and impaired stress recovery exclusively in individuals with access to visual social stimuli. Our findings contribute to the growing body of evidence that social stimuli buffer stress responses in fish and suggest that oxytocin receptors may play a role in socially-buffered stress recovery across taxa.

Elder mistreatment and psychological resilience: the moderating role of social support.

This study examines the possible consequence of elder mistreatment on resilience and whether social support moderates this posited association, using a rural sample of 432 community-dwelling older Chinese adults aged 60 to 79 years. Elder abuse included verbal abuse, physical abuse, or financial exploitation. Social support was measured by The Multidimensional Scale of Perceived Social Support (MSPSS). Resilience was represented by a seven-item scale. Hierarchical regression models indicated that mistreatment is significantly related to low levels of resilience when confounding factors are adjusted. A significant interaction term (abuse × social support) was also detected. Mistreated respondents who reported higher levels of social support were less likely to experience low resilience compared to those with lower levels of social support. Social support buffers against the undesirable effect of mistreatment on resilience, especially for those who were abused.

Mitigating the affective and cognitive consequences of social exclusion: an integrative data analysis of seven social disconnection interventions.

BACKGROUND: Being socially excluded has detrimental effects, with prolonged exclusion linked to loneliness and social isolation. Social disconnection interventions that do not require direct support actions (e.g., "how can I help?") offer promise in mitigating the affective and cognitive consequences of social exclusion. We examine how various social disconnection interventions involving friends and unknown peers might mitigate social exclusion by buffering (intervening before) and by promoting recovery (intervening after). METHODS: We present an integrative data analysis (IDA) of five studies (N = 664) that systematically exposed participants to exclusion (vs. inclusion) social dynamics. Using a well-validated paradigm, participants had a virtual interaction with two other people. Unbeknownst to participants, the other people's behavior was programmed to either behave inclusively toward the participant or for one to behave exclusively. Critically, our social disconnection interventions experimentally manipulated whether a friend was present (vs. an unknown peer vs. being alone), the nature of interpersonal engagement (having a face-to-face conversation vs. a reminder of an upcoming interaction vs. mere presence), and the timing of the intervention in relation to the social dynamic (before vs. during vs. after). We then assessed participants' in-the-moment affective and cognitive responses, which included mood, feelings of belonging, sense of control, and social comfort. RESULTS: Experiencing exclusion (vs. inclusion) led to negative affective and cognitive consequences. However, engaging in a face-to-face conversation with a friend before the exclusion lessened its impact (p < .001). Moreover, a face-to-face conversation with a friend after exclusion, and even a reminder of an upcoming interaction with a friend, sped-up recovery (ps < .001). There was less conclusive evidence that a face-to-face conversation with an unknown peer, or that the mere presence of a friend or unknown peer, conferred protective benefits. CONCLUSIONS: The findings provide support for the effectiveness of social disconnection interventions that involve actual (i.e., face-to-face) or symbolic (i.e., reminders) interactions with friends. These interventions target momentary vulnerabilities that arise from social exclusion by addressing negative affect and cognitions before or after they emerge. As such, they offer a promising approach to primary prevention prior to the onset of loneliness and social isolation.

Maternal caregiving stress and metabolic health: Sexual activity as a potential buffer.

Chronic stress lead to dysregulation of metabolic hormones, creating risk for obesity and type 2 diabetes. Based on previous work suggesting the potential for sexual activity to relieve psychological stress and reduce stress-related neuroendocrine activity, the present research explored sexual activity as a protective factor. We focused on chronic stress in the form of caregiving stress, comparing premenopausal mothers of a child with an autism spectrum disorder vs. a neurotypical child, in relation to metabolic hormones - insulin (and insulin resistance as assessed by HOMA), leptin, and ghrelin. Then, we explored the moderating role of sexual activity. Our results showed that high-stress mothers showed higher levels of insulin, insulin resistance, and lower levels of ghrelin compared to low-stress mothers. However, sexual activity modulated these associations such that among mothers who were sexually active (as coded from their daily diaries), no significant differences in these outcomes were observed between groups. This buffering effect of sexual activity was distinguishable from the buffering effect of physical activity and independent of global relationship satisfaction. Together, our findings provide novel evidence supporting the potential protective effects of sexual activity from chronic stress-related metabolic disease risk.

Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies.

Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.

Mechanisms of whole body, respiratory, acid-base buffering: a first computer-model test of three physicochemical, acid-base theories.

Acid-base disorders are currently analyzed and treated using a bicarbonate-centered approach derived from blood studies prior to the advent of digital computers, which could solve computer models capable of quantifying the complex physicochemical nature governing distribution of water and ions between fluid compartments. An alternative is the Stewart approach, which can predict the pH of a simple mixture of ions and electrically charged proteins; hence, the role of extravascular fluids has been largely ignored. The present study uses a new, comprehensive computer model of four major fluid compartments, based on a recent blood model, which included ion binding to proteins, electroneutrality constraints, and other essential physicochemical laws. The present model predicts quantitative respiratory acid-base buffering behavior in the whole body, as well as determining roles of each compartment and their species, particularly compartmental electrically charged proteins, largely responsible for buffering. The model tested an early theory that H+ was conserved in the body fluids; hence, when changing Pco2 states, intracellular buffering could be predicted by net changes in bicarbonate and protein electrical charge in the remaining fluids. Even though H+ is not conserved in the model, the theory held in simulated respiratory disorders. Model results also agreed with a second part of the theory, that ion movements between cells and interstitial fluid were linked with H+ buffering, but by electroneutrality constraints, not necessarily by some membrane-related mechanisms, and that the strong ion difference (SID), an amalgamation of ionic electrical charges, was approximately conserved when going between equilibrium states caused by Pco2 changes in the body-fluid system.NEW & NOTEWORTHY For the first time, a physicochemically based, whole body, four-compartment, computer model was used to study respiratory whole body acid-base buffering. An improved approach to quantify acid-base buffering, previously used by this author, was able to determine contributions of the various compartmental fluids to whole body buffering. The model was used to test, for the first time, three fundamental theories of whole body acid-base homeostasis, namely, H+-conservation, its linkage to ion transport, and strong ion difference conservation.

Mutational robustness and the role of buffer genes in evolvability.

Organisms rely on mutations to fuel adaptive evolution. However, many mutations impose a negative effect on fitness. Cells may have therefore evolved mechanisms that affect the phenotypic effects of mutations, thus conferring mutational robustness. Specifically, so-called buffer genes are hypothesized to interact directly or indirectly with genetic variation and reduce its effect on fitness. Environmental or genetic perturbations can change the interaction between buffer genes and genetic variation, thereby unmasking the genetic variation's phenotypic effects and thus providing a source of variation for natural selection to act on. This review provides an overview of our understanding of mutational robustness and buffer genes, with the chaperone gene HSP90 as a key example. It discusses whether buffer genes merely affect standing variation or also interact with de novo mutations, how mutational robustness could influence evolution, and whether mutational robustness might be an evolved trait or rather a mere side-effect of complex genetic interactions.

The impact of widowhood on the mental health of older adults and the buffering effect of social capital.

Background: Widowhood is one of the most serious issues affecting the mental health of older persons. China currently has tens of millions of widowed older adult, which is a huge group. It is of great significance to study the impacts of widowhood on their mental health and put forward some measures for improvement. Method: We used China Family Panel Studies (CFPS) data in 2020, which included 4,184 older adults. Linear regression is used to examine the relationship among widowhood, mental health, and social capital. Results: Both short-term and medium- and long-term widowhood lead to a significant increase in depression, which seriously affects the mental health of older people. At the same time, community-level and family-level social capital have significant buffering effects on the loss of mental health caused by widowhood, but this effect is heterogeneous, with different types of social capital playing different roles among different gender groups. Conclusion: The provision of care support by children and good neighborhood relationships can help mitigate the psychological impact of widowhood, and these are areas where social policy can make a difference.

Paternal and Maternal Depressive Symptoms and Sensitivity: Links with Trajectories of Socioemotional Problems in Toddlerhood.

Parental depressive symptoms and sensitivity have well-documented consequences for children; however, studies considering both parents are still scarce. This longitudinal study aimed to investigate the respective roles of paternal and maternal depressive symptoms and sensitivity in predicting the development of child socioemotional problems during toddlerhood. We also investigated the buffering role of each parent's sensitivity in the associations between the other parent's depressive symptoms and toddlers' socioemotional problems. The sample consisted of 140 Canadian families who were visited in their homes when children were around 13 (T1), 19 (T2), and 27 (T3) months of age. At T1, both parents' sensitivity was assessed from observations of parent-child interactions at home and each parent reported on his or her own depressive symptoms. At T1, T2, and T3, maternal and paternal perceptions of their toddler's socioemotional problems were assessed and aggregated. Growth curve analyses revealed that paternal and maternal depressive symptoms as well as paternal sensitivity were unique and persistent predictors of child socioemotional problems and that sensitive fathering acted as a buffer in the context of maternal depressive symptoms. This study highlights the importance of considering both parents when studying risk and protective factors for young children's socioemotional problems.

Purpose in life moderates the relationship between loneliness and caregiving stress among family caregivers of people with mental health problems.

OBJECTIVE: Providing care to others can exert a profound impact on caregivers' sense of purpose or meaning in life, thereby reciprocally influencing the caregivers' overall health and well-being. This study aims to investigate whether the sense of purpose in life moderates the association between loneliness and caregiving stress among family caregivers of people with mental health problems. METHODS: A sample of family caregivers of people with mental health problems (N = 468, 57.1 % female) drawn from the 2020 survey of the Caregiving in the U.S. was investigated. Descriptive statistics, correlation analysis, and a multiple regression with an interaction term were performed. RESULTS: Higher levels of loneliness were associated with enhanced caregiving stress. Moreover, after demographic and care-related factors were controlled for, the association between loneliness and caregiving stress was moderated by purpose in life; namely, as the sense of purpose in life increased, so did the intensity of the relationship between loneliness and caregiving stress. CONCLUSION: Reducing loneliness or strengthening the sense of purpose helps alleviate caregiving stress, and lonely family caregivers with a strong sense of purpose deserve extra attention.

Role of protein kinase A and A kinase anchoring proteins in buffering and compartmentation of cAMP signalling in human airway smooth muscle cells.

BACKGROUND AND PURPOSE: In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor-mediated, compartmentalized responses. EXPERIMENTAL APPROACH: Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA-AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down-regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors. KEY RESULTS: Cyclic AMP movement was significantly slower than that of free-diffusion in hASM cells, and disrupting PKA-AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D-AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor-mediated responses. CONCLUSION AND IMPLICATIONS: Our results confirm that AKAP-anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells.