Regulation of Piezo Channels by Cellular Signaling Pathways.

The recently identified mechanically activated Piezo1 and Piezo2 channels play major roles in various aspects of mechanosensation in mammals, and their mutations are associated with human diseases. Recent reports show that activation of cell surface receptors coupled to heterotrimeric Gq proteins increase the sensitivity of Piezo2 channels to mechanical stimuli. Activation of the cyclic adenosine monophosphate pathway was also shown to potentiate Piezo2 channel activity. This phenomenon may play a role in mechanical allodynia or hyperalgesia during inflammation. Both Piezo1 and Piezo2 channels are inhibited upon depletion of plasma membrane phosphoinositides, in response to phospholipase C activation by Ca2+ influx via the transient receptor potential vanilloid 1 channels. This review will discuss current knowledge on regulation of Piezo channels by these intracellular signaling pathways.

Stimulus-secretion coupling and mitochondrial metabolism in steroid-secreting cells.

Ca(2+) signal in high-Ca(2+) perimitochondrial microdomains is amplified within the mitochondrial matrix and activates Ca(2+)-dependent dehydrogenases. In steroid-secreting cells, small cytoplasmic Ca(2+) signals may also augment mitochondrial Ca(2+) concentration. The ensuing formation of NADH and NADPH may have an essential role in supporting the increased steroid secretion.

Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides.

Activation of several inwardly rectifying K(+) channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P(2) strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P(2). We investigated whether these inwardly rectifying K(+) channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). In contrast, IRK1 channels were not activated by PtdIns(3,4)P(2) and only marginally by high concentrations of PtdIns(3,5)P(2). Similarly, high concentrations of PtdIns(3,4,5)P(3) were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P(2), whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P(2) and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P(2). Incorporation of PtdInsP(2) into the membrane is necessary for activation, as the short chain water soluble diC(4) PtdIns(4,5)P(2) did not activate either channel, whereas activation by diC(8) PtdIns(4, 5)P(2) required high concentrations.

Cytoplasmic Ca2+ signalling and reduction of mitochondrial pyridine nucleotides in adrenal glomerulosa cells in response to K+, angiotensin II and vasopressin.

We have examined the mitochondrial formation of NAD(P)H in rat adrenal glomerulosa cells. A short-term elevation of the K+ concentration from 3.6 to 8.4 mM induced a reversible increase in the formation of reduced pyridine nucleotides. Potassium applied after the addition of rotenone had no further effect, confirming that the redox signal was of mitochondrial origin. Inhibition of aldosterone synthesis by aminoglutethimide in K+-stimulated cells decreased the rate of decay of the NAD(P)H signal upon the termination of stimulation, indicating that the NADPH formed was consumed in aldosterone synthesis. When the NAD(P)H signal was measured simultaneously with the cytoplasmic free Ca2+ concentration ([Ca2+]i), elevation of the K+ concentration to 6.6 or 8.4 mM induced parallel increases in [Ca2+]i and NAD(P)H formation. The rates of increase and decrease of NAD(P)H were lower than for [Ca2+]i, confirming that the redox signal was secondary to the Ca2+ signal. Angiotensin II (100 pM-1 nM) induced an oscillatory NAD(P)H signal which usually returned to a lower baseline concentration, while a sustained signal with superimposed oscillations was observed at higher concentrations. Simultaneous measurements showed that NAD(P)H levels followed the [Ca2+]i pattern evoked by angiotensin II. Vasopressin (100 nM) also induced parallel oscillations of [Ca2+]i and NAD(P)H. A sustained rise in the extramitochondrial Ca2+ concentration to 1 microM induced a sustained elevation of the intramitochondrial Ca2+ concentration in permeabilized cells, as measured with rhod-2. A sustained rise in [Ca2+]i evoked by long-term stimulation with 8.4 mM K+ or 2.5 nM angiotensin II resulted in sustained NAD(P)H production. These Ca2+-dependent changes in the mitochondrial redox state support the biological response, i.e. aldosterone secretion by glomerulosa cells.

Intracellular calcium release is more efficient than calcium influx in stimulating mitochondrial NAD(P)H formation in adrenal glomerulosa cells.

We compared the effect on mitochondrial NAD(P)H formation of calcium release from intracellular stores with that of calcium influx from the extracellular space. Simultaneous measurements of cytoplasmic free calcium ion concentration and mitochondrial NAD(P)H were performed on fura-PE3-loaded single rat adrenal glomerulosa cells. The effects of equipotent stimuli in terms of the evoked Ca2+ response were compared. Angiotensin II (AII; 1 nM) induced a higher amplitude NAD(P)H response than K+ (5.6-7.6 mM). Vasopressin (1 microM) also induced a greater initial NAD(P)H formation than K+, although the Ca2+ signal evoked by the two agonists had similar amplitude. To examine the effect of Ca2+ release from internal stores we applied AII in Ca2+-free medium. We compared the effect on NAD(P)H formation of Ca2+ release with Ca2+ influx induced by K+, and with capacitative Ca2+ influx induced by AII. NAD(P)H formation in response to Ca2+ release was greater than that induced by Ca2+ influx, irrespective of whether induced by K+ or AII. Our results indicate that Ca2+, presumably released in the vicinity of mitochondria, activates mitochondrial dehydrogenases more efficiently than Ca2+ entering through the plasma membrane. These data confirm the biological significance of previous observations showing that Ca2+ released from inositol 1,4, 5-trisphosphate-sensitive internal stores increases mitochondrial matrix [Ca2+] to a greater extent than extracellular Ca2+.

The role of voltage-dependent calcium channels in angiotensin-stimulated glomerulosa cells.

The concept that voltage-dependent Ca2+ influx is essential in the aldosterone stimulating action of angiotensin II (AII) has been recently challenged by the demonstration of the dihydropyridine (DHP) insensitive 'capacitative' Ca2+ uptake mechanism. The DHP-sensitivity of AII-induced aldosterone secretion is still to be explained. In rat glomerulosa cells the lag phase of AII-induced depolarization is more than 30 s, and there is no enhanced Ca2+ influx within the first min of stimulation. Yet we observed that DHPs as well as diltiazem influenced also the peak of cytoplasmic Ca2+ signal, although the peak (approximately 12 s) is attributed to Ca2+ release alone. Nifedipine reduced the Ca2+ transient induced by AII even after complete inhibition of Ca2+ channel activity. Recalling the loose attachment of InsP3 receptors (IP3R) to the plasma membrane, and the homology between the cytosolic domain of IP3R and the Ca2+ release channel (ryanodine receptor) of skeletal muscle, we proposed that DHP-sensitive L-type Ca2+ channels (DHP receptors) influence InsP3-induced Ca2+ release rather than Ca2+ influx in AII-stimulated cells. Although the dominant isoform is the neuroendocrine (D) one, the skeletal muscle isoform of L-type voltage-dependent Ca2+ channel is also expressed in rat glomerulosa cells. This isoform may be a candidate for protein-protein interaction between DHPR and subplasmalemmal IP3R, similarly to that occurring between DHP receptors and ryanodine receptors in skeletal muscle.

Expression of inositol 1,4,5-trisphosphate receptors in rat adrenocortical zones.

Previously we demonstrated the presence of InsP3R-I, -II and -III subtypes in the zona glomerulosa. Now we have examined the expression of different subtypes of inositol 1,4,5-trisphosphate receptor (InsP3R) in the inner zones of rat adrenal cortex. RNA extracted from decapsulated adrenal tissue (zonae fasciculata-reticularis and the medulla) or from fasciculata-reticularis cells was reverse transcribed. Subsequent polymerase chain reaction revealed the presence of InsP3R-I, -II and -III subtypes in decapsulated tissue but failed to demonstrate the expression of any known subtypes of InsP3R in fasciculata-reticularis cells. Accordingly, InsP3 receptors expressed in the decapsulated tissue are of medullary origin.

Capacitative Ca2+ influx in adrenal glomerulosa cells: possible role in angiotensin II response.

We examined the effect of the depletion of intracellular Ca2+ stores on Ca2+ influx in rat glomerulosa cells. Depletion of intracellular Ca2+ stores was achieved by inhibiting sarco/endoplasmic reticulumtype Ca(2+)-ATPase with thapsigargin or 2,5,di-(t-butyl)-1,4-benzohydroquinone (t-BHQ). Both inhibitors induced a sustained rise in cytoplasmic Ca2+ concentration. The initial rise was observed also in Ca(2+)-free medium, while the sustained phase disappeared, indicating that the latter requires Ca2+ influx. In Ca(2+)-free medium, the readdition of Ca2+ induced a steeper and higher rise in intracellular Ca2+ concentration in thapsigargin-treated cells than in controls, supporting the role of Ca2+ influx. In normal medium, the addition of Cd2+ (80 microM) evoked an immediate inhibition of the sustained phase of thapsigargin response. The response to thapsigargin was insensitive to nifedipine. Thapsigargin failed to enhance Mn2+ quenching of fura 2. Our results provide evidence for the existence of capacitative Ca2+ influx in rat glomerulosa cells and indicate that dihydropyridine-sensitive Ca2+ channels do not participate in capacitative Ca2+ entry. High concentrations of thapsigargin and t-BHQ, similar to the reported effects of angiotensin II and vasopressin, inhibited K(+)-induced Ca2+ signals. These effects appear, however, to be independent of the depletion of internal Ca2+ stores.

Plasmalemmal dihydropyridine receptors modify the function of subplasmalemmal inositol 1,4,5-trisphosphate receptors: a hypothesis.

Experimental observations on rat glomerulosa cells inspired a model which postulates that plasmalemmal dihydropyridine receptors are in juxtaposition and interaction with inositol 1,4,5-trisphosphate receptors in subplasmalemmal calciosomes. Activation of dihydropyridine receptors promotes the Ca2+ releasing effect of inositol 1,4,5-trisphosphate. The most important observations compatible with the model are the following: (1) angiotensin II does not influence Ca2+ influx during the peak phase of Ca2+ signal; (2) dihydropyridine drugs modify the initial peak of the Ca2+ signal induced by angiotensin II; (3) inhibitors of the dihydropyridine receptor reduce the initial Ca2+ signal also in the presence of 5 mM Ni2+, an inhibitor of voltage dependent Ca2+ influx; and (4) changes in extracellular K+ concentration within the physiological range also modify the cytoplasmic Ca2+ response to angiotensin II.

Dihydropyridine-sensitive initial component of the ANG II-induced Ca2+ response in rat adrenal glomerulosa cells.

The Ca2+ signal induced by an increase in extracellular K+ concentration from 3.6 to 5.6 mM or angiotensin II (ANG II) was inhibited by the dihydropyridine (DHP) Ca2+ channel blocker, nifedipine, and enhanced by the DHP Ca2+ channel agonist, BAY K 8644. The DHP sensitivity of the ANG II-induced Ca2+ response was already detectable during the peak phase, suggesting that the DHP receptor plays an important role during the initial phase of ANG II stimulation. K+ and ANG II stimulated a nifedipine-sensitive Mn2+ influx pathway, further promoting the role of a DHP receptor in their mechanism of action. Fluorescent membrane potential measurements showed that, in contrast to the rapid depolarization induced by K+, the ANG II-induced depolarization had a lag time of > 30 s. The slow kinetics of depolarization compared with the immediate effect of ANG II on Mn2+ influx and the DHP sensitivity of the initial Ca2+ peak indicates that ANG II initiates the activation of the DHP-sensitive Ca2+ channel by a mechanism other than depolarization.

Sustained stimulation of aldosterone production by angiotensin II is potentiated by nickel.

Angiotensin-induced aldosterone production by superfused adrenal glomerulosa cells was potentiated by Ni2+ (0.1 mM), added either at the onset of stimulation with angiotensin II or 1 h later. Nickel did not influence the effect of adrenocorticotropic hormone or potassium on aldosterone production. Nickel failed to modify angiotensin-induced changes in phospholipid metabolism or the formation of inositol phosphates and slightly reduced the enhancement of 45Ca influx. Uptake of Ni2+ into glomerulosa cells was increased by depolarization in a dihydropyridine-insensitive manner. Because nickel selectively potentiates the sustained phase of the response to a calcium-mobilizing hormone, it may serve as a suitable tool in elucidating the signal transduction process during the sustained phase of stimulation.