Collecting duct water permeability inhibition by EGF is associated with decreased cAMP, PKA activity, and AQP2 phosphorylation at Ser269.

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-d-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser264 and Ser269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at Ser256, Ser264, and Ser269. None of the other kinases tested phosphorylated Ser269. In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269. These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269, a site previously shown to regulate AQP2 endocytosis.NEW & NOTEWORTHY The authors used native rat collecting ducts to show that inhibition of vasopressin-stimulated water permeability by epidermal growth factor involves a reduction of aquaporin 2 phosphorylation at Ser269, a consequence of reduced cAMP production and PKA activity.

PIEZO2-dependent rapid pain system in humans and mice.

The PIEZO2 ion channel is critical for transducing light touch into neural signals but is not considered necessary for transducing acute pain in humans. Here, we discovered an exception - a form of mechanical pain evoked by hair pulling. Based on observations in a rare group of individuals with PIEZO2 deficiency syndrome, we demonstrated that hair-pull pain is dependent on PIEZO2 transduction. Studies in control participants showed that hair-pull pain triggered a distinct nocifensive response, including a nociceptive reflex. Observations in rare Aß deafferented individuals and nerve conduction block studies in control participants revealed that hair-pull pain perception is dependent on Aß input. Single-unit axonal recordings revealed that a class of cooling-responsive myelinated nociceptors in human skin is selectively tuned to painful hair-pull stimuli. Further, we pharmacologically mapped these nociceptors to a specific transcriptomic class. Finally, using functional imaging in mice, we demonstrated that in a homologous nociceptor, Piezo2 is necessary for high-sensitivity, robust activation by hair-pull stimuli. Together, we have demonstrated that hair-pulling evokes a distinct type of pain with conserved behavioral, neural, and molecular features across humans and mice.

Human-Delivered Brushstroke Characterization using an Instrumented Brush Focused on Torque.

Pleasant brush therapies may benefit those with autism, trauma, and anxiety. While studies monitor brushing velocity, hand-delivery of brush strokes introduces variability. Detailed measurements of human-delivered brushing physics may help understand such variability and subsequent impact on receivers' perceived pleasantness. Herein, we instrument a brush with multi-axis force and displacement sensors to measure their physics as 12 participants pleasantly stroke a receiver's forearm. Algorithmic procedures identify skin contact, and define four stages of arrival, stroke, departure, and airtime between strokes. Torque magnitude, rather than force, is evaluated as a metric to minimize inertial noise, as it registers brush bend and orientation. Overall, the results of the naturally delivered brushing experiments indicate force and velocity values in the range of 0.4 N and 3-10 cm/s, in alignment with prior work. However, we observe significant variance between brushers across velocity, force, torque, and brushstroke length. Upon further analysis, torque and force measures are correlated, yet torque provides distinct information from velocity. In evaluating the receiver's response to individual differences between brushers of the preliminary case study, higher pleasantness is tied to lower mean torque, and lower instantaneous variance over the stroke duration. Torque magnitude appears to complement velocity's influence on perceived pleasantness.

Optical Measurements of the Skin Surface to Infer Bilateral Distinctions in Myofascial Tissue Stiffness.

About half the U.S. adult population suffers from chronic neuromusculoskeletal pain. While its evaluation and treatment are widely addressed by therapies using soft tissue manipulation (STM), their efficacy is based upon clinician judgment. Robust biomarkers are needed to quantify the effects of STM on patient outcomes. Among noninvasive methods to quantify the mechanics of myofascial tissue, most are limited to small (<10 mm2), localized regions of interest. In contrast, we develop an approach to optically simultaneously measure a larger (~100 cm2) field of deformation at the skin surface. Biomarkers based on skin lateral mobility are derived to infer distinctions in myofascial tissue stiffness. In specific, three cameras track ink speckles whose fields of deformation and stretch are resolved with digital image correlation. Their ability to differentiate bilateral distinctions of the cervicothoracic region is evaluated with four participants, as a licensed clinician performs STM. The results indicate that the optically derived surface biomarkers can differentiate bilateral differences in skin mobility, with trend directions within a participant similar to measurements with an instrumented force probe. These findings preliminarily suggest skin surface measurements are capable of inferring underlying myofascial tissue stiffness, although further confirmation will require a larger, more diverse group of participants.

Using Digital Image Correlation to Quantify Skin Deformation With Von Frey Monofilaments.

Thin von Frey monofilaments are a clinical tool used worldwide to assess touch deficits. One's ability to perceive touch with low-force monofilaments (0.008 - 0.07 g) establishes an absolute threshold and thereby the extent of impairment. While individual monofilaments bend at defined forces, there are no empirical measurements of the skin surface's response. In this work, we measure skin surface deformation at light-touch perceptual limits, by adopting an imaging approach using 3D digital image correlation (DIC). Generating point cloud data from three cameras surveilling the index finger pad, we reassemble and stitch together multiple 3D surfaces. Then, in response to each monofilament's indentation over time, we quantify strain across the skin surface, radial deformation emanating from the contact point, penetration depth into the surface, and area between 2D cross-sections. The results show that the monofilaments create distinct states of skin deformation, which align closely with just noticeable percepts at absolute detection and discrimination thresholds, even amidst variance between individuals and trials. In particular, the resolution of the DIC imaging approach captures sufficient differences in skin deformation at threshold, offering promise in understanding the skin's role in perception.

Phosphoproteomic identification of vasopressin V2 receptor-dependent signaling in the renal collecting duct.

Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.