Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1.

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca2+) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd3+). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA.

TRPV channels and vascular function.

Transient receptor potential channels, of the vanilloid subtype (TRPV), act as sensory mediators, being activated by endogenous ligands, heat, mechanical and osmotic stress. Within the vasculature, TRPV channels are expressed in smooth muscle cells, endothelial cells, as well as in peri-vascular nerves. Their varied distribution and polymodal activation properties make them ideally suited to a role in modulating vascular function, perceiving and responding to local environmental changes. In endothelial cells, TRPV1 is activated by endocannabinoids, TRPV3 by dietary agonists and TRPV4 by shear stress, epoxyeicosatrienoic acids (EETs) and downstream of Gq-coupled receptor activation. Upon activation, these channels contribute to vasodilation via nitric oxide, prostacyclin and intermediate/small conductance potassium channel-dependent pathways. In smooth muscle, TRPV4 is activated by endothelial-derived EETs, leading to large conductance potassium channel activation and smooth muscle hyperpolarization. Conversely, smooth muscle TRPV2 channels contribute to global calcium entry and may aid constriction. TRPV1 and TRPV4 are expressed in sensory nerves and can cause vasodilation through calcitonin gene-related peptide and substance P release as well as mediating vascular function via the baroreceptor reflex (TRPV1) or via increasing sympathetic outflow during osmotic stress (TRPV4). Thus, TRPV channels play important roles in the regulation of normal and pathological cellular function in the vasculature.

Inhibition of the cardiac L-type calcium channel current by the TRPM8 agonist, (-)-menthol.

(-)-Menthol and icilin are agonists of the thermoreceptor non-selective cation channel, TRPM8, and are commonly used to investigate TRPM8 function without a full appreciation of their non-specific effects. To investigate the hypothesis that (-)-menthol and icilin inhibit cardiovascular-type L-type Ca(2+) channel currents (I(Ca,L)), the actions of the TRPM8 agonists on rabbit ventricular myocyte I(Ca,L) were examined at near-physiological temperature (≈35°C) using whole-cell recording. Icilin (3-100 µM) did not significantly inhibit I(Ca,L). (3) in contrast, (-)-menthol concentration-dependently inhibited peak I(Ca,L) (IC(50)=74.6 µM; log(10)IC(50)(M)=-4.13±0.14). (-)-Menthol blocked the late I(Ca,L) remaining at the end of depolarising pulses with greater efficacy (96.1±2.4% block at 1 mM) than peak I(Ca,L) (68.9±5.7% block at 1 mM, P<0.01), although there was no difference in potency of block of peak and late currents. Block by (-)-menthol showed no voltage-dependence. The actions of (-)-menthol were compared with those of nimodipine. Nimodipine was a more efficacious (97.3±1.5 % block at 30 µM, P<0.01) and potent (IC(50)=0.74 µM; log(10)IC(50)(M)=-6.13±0.08, P<0.0001) blocker of peak I(Ca,L) than (-)-menthol. In contrast to (-)-menthol, nimodipine showed greater potency (IC(50)=0.056 µM; log(10)IC(50)(M)=-7.25±0.17, P<0.0001), but not greater efficacy, in block of late compared with peak I(Ca,L). In summary, these data demonstrate that, at near-physiological temperature, (-) -menthol blocks cardiac I(Ca,L) at concentrations similar to those reportedly effective in TRPM8-agonism. The data suggest that the mechanism of L-type Ca(2+) channel block by (-)-menthol differs from that of nimodipine.