Piezo1 integration of vascular architecture with physiological force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic¹⁻⁵. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca²⁺-permeable non-selective cationic channels for detection of noxious mechanical impact⁶⁻⁸. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology

Purinergic-mediated Ca2+ influx in Dictyostelium discoideum

The presence of five P2X-like genes (p2xA–E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca2+ influx in response to extracellular ATP and ADP (EC50 = 7.5 μM and 6.1 μM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91 ± 0.04 s, required extracellular Ca2+, were inhibited by Gd3+, modified by extracellular pH and were not affected by deletion of either the single Gβ or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu2+ and Zn2+ inhibited purine-evoked Ca2+ influx with IC50 values of 0.9 and 6.3 μM, respectively. 300 μM Zn2+ completely abolished the initial large rapid rise in intracellular Ca2+ revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling

7α-methyl-19-nortestosterone (MENT) vs testosterone in combination with etonogestrel implants for spermatogenic suppression in healthy men

Testosterone with a progestogen can suppress spermatogenesis for contraception. The synthetic androgen 7α-methyl-19-nortestosterone (MENT) may offer advantages because it is resistant to 5α-reduction and is therefore less active at the prostate. This study aimed to investigate MENT implants in combination with etonogestrel on spermatogenesis, gonadotropins, and androgen-dependent tissues in comparison with a testosterone/etonogestrel regimen. Healthy men (n = 29) were recruited and randomized to receive 2 etonogestrel implants with either 600-mg testosterone pellets repeated every 12 weeks or 2 MENT implants for up to 48 weeks. Testosterone concentrations in the testosterone group remained in the normal range. Subjects with 2 MENT implants showed peak MENT levels at 4 weeks with testosterone concentrations of 2 nmol/L. Sperm concentrations fell rapidly to less than 1 × 106/mL at 12 weeks in 8 of 10 subjects in the MENT group and 13 of 16 subjects in the testosterone group with equally suppressed gonadotropins. Thereafter, suppression was not maintained in the MENT group, and 6 men noted loss of libido. Fourteen men completed 48 weeks of testosterone treatment, and all became azoospermic. Hemoglobin concentrations rose, and high density lipoprotein-cholesterol (HDL-C) fell in both groups. The MENT group showed a fall in prostate-specific antigen with no change in bone mass. MENT with a progestogen can achieve rapid suppression of spermatogenesis similar to testosterone, but this promising result was not sustained due to a decline in MENT release from the implants. This dose of testosterone, compared with previous studies using a lower dose with a higher dose of etonogestrel, had nonreproductive side effects without any increase in spermatogenic suppression. These data indicate the importance of the doses of progestogen and testosterone for optimum spermatogenic suppression while minimizing side effects

Extracellular ATP and ADP mediate Ca2+ influx in Dictyostelium discoideum.

Extracellular ATP and ADP mediate Ca2+ influx in Dictyostelium discoideum

Clathrin-independent hERG internalisation.

<p>(A) The distribution of HA-hERG_hERG or HA-K_ATP transiently expressed in HeLa cells was compared after 30 minutes incubation with anti-HA in the presence or absence of 80 µM dynasore, 5 mM MβCD, 5 mM αCD or 15 µM/50 µM SecinH3. Cells were pre-incubated with each drug for 30 minutes. After fixation total HA-hERG_hERG or HA-K_ATP were stained with anti-Kv11.1 (recognising a C-terminal epitope) or anti-SUR1 respectively. Bar = 20 µm. (B-C) Internalisation of HA-hERG_hERG was quantified in HEK-MSR cells. After 30 minutes at 37°C with anti-HA cells were fixed, incubated with HRP-conjugated secondary antibodies and exposed to TMB substrate. Internalisation was considered as a ratio of surface (non-permeabilised cells) to surface + internalised (permeabilised cells). Increases in this ratio, indicative of decreased internalisation, were probed for after treatment with 80 µM dynasore and 15 µM SecinH3 (B) (n = 6) or co-transfection with dominant negative Rab5 and Arf6 expression vectors (C) (n = 4). </p

Surface density of HA-hERG containing channels.

<p>(A) Schematic of the transmembrane topology of the hERG subunit illustrating the insertion site of the HA sequence. (B) HEK-MSR cells were transfected with various ratios of HA-hERG and hERG expression vectors. Surface (non-permeabilised) and total (permeabilised) levels of HA-hERG_hERG were quantified using TMB substrate (n ≥ 4).</p

Clathrin-independent internalisation in myocytes.

<p>Effects of 80 µM dynasore, 3 mM MβCD, 3 mM αCD and 50 µM SecinH3 on internalisation of native ERG in NRVCM at 37°C. Cells were incubated for 1 hour with anti-Kv11.1 (extracellular epitope), fixed and stained with Cy3-conjugated secondary antibodies. In the bottom row, cells were washed with acidic buffer prior to fixation to remove surface bound antibodies. Staining of nuclei (inset) confirms the presence of cells within the field of view for 4°C and MβCD. Bar = 10 µm.</p

Internalised hERG channels and transferrin predominantly localise to distinct endosomes.

<p>HeLa cells transfected with HA-hERG_hERG were incubated with anti-HA prior to washing, fixing, permeabilisation and staining with Cy3 conjugated secondary antibody. Alexa Fluor^® 488-conjugated Tfn was used as marker of the Tfn receptor. Different incubation times (indicated in minutes in bottom left corner of images) at 37°C were used to compare protein distributions following 5 minutes internalisation (A) or after allowing sufficient time to label recycling pathways (C). Incubation at 16°C (B) inhibits trafficking of the Tfn receptor beyond EE, facilitating assessment of early trafficking events. Amplified boxes highlight points of co-localisation and bars = 10 µm. </p

Arf6-T27N increases hERG current density.

<p>Assessment of dominant negative Arf6, cdc42 and µ2 constructs on hERG current density. (A) Current-voltage relationships of hERG channels co-expressed with Arf6-T27N-eGFP, eGFP-cdc42-T17N or µ2-D176A/W421A in HEK-MSR cells; representative current families are shown. (B) Current-voltage relationships of tail currents determined as in (A) (n = 4-9). (C) Current densities measured from tail currents at -60 mV after a +60 mV prepulse (data from (B)) (n = 4-9).</p

Arf6-Q67L-eGFP positive vacuoles contain hERG channels.

<p>hERG channels expressed in HeLa (A) or H9c2 cells (B&C) were stained with anti-Kv11.1 and Cy3-conjugated secondary antibodies and their distribution compared with co-expressed Arf6-Q67L-eGFP (A&C). In H9c2 cells Arf6-Q67L-eGFP (C) causes an increased central localisation of hERG compared with control (B). Amplified boxes highlight points of co-localisation and bars = 10 µm. </p