Jens Leipziger

ATP release from non-excitable cells

All cells release nucleotides and are in one way or another involved in local autocrine and paracrine regulation of organ function via stimulation of purinergic receptors. Significant technical advances have been made in recent years to quantify more precisely resting and stimulated adenosine triphosphate (ATP) concentrations in close proximity to the plasma membrane. These technical advances are reviewed here. However, the mechanisms by which cells release ATP continue to be enigmatic. The current state of knowledge on different suggested mechanisms is also reviewed. Current evidence suggests that two separate regulated modes of ATP release co-exist in non-excitable cells: (1) a conductive pore which in several systems has been found to be the channel pannexin 1 and (2) vesicular release. Modes of stimulation of ATP release are reviewed and indicate that both subtle mechanical stimulation and agonist-triggered release play pivotal roles. The mechano-sensor for ATP release is not yet defined.

Autocrine Purinergic Receptor Signaling Is Essential for Macrophage Chemotaxis

Amplification of outside-in chemotactic signaling by inside-out purinergic signaling drives macrophage migration. Self-Help Migration Immune cells such as neutrophils and macrophages migrate to sites of infection or inflammation by following gradients of chemoattractants. These include chemokines, which can be released by other cells at the target site; components of the complement system, such as C5a; and bacterial products, such as the formylated peptide, fMLP. While they navigate along the chemoattractant gradient toward their destination, cells are also exposed to other signals, some of which may compete with the chemoattractant that the cells were already following. Another level of complexity in the regulation of cell migration came from the discovery that migrating neutrophils release adenosine triphosphate (ATP), which then functions in an autocrine fashion through the purinergic receptor P2Y2 to enhance migration; cells deficient in P2Y2 have impaired gradient sensing. Kronlage et al. provide evidence that autocrine ATP signaling is also required for the migration of macrophages in vitro and in an in vivo model. The authors found that more than one type of ATP receptor type as well as metabolites of ATP contributed to the migratory responses of macrophages; furthermore, ATP was not released through pannexin-1 proteins, as has been suggested for neutrophils. Together, these data suggest that autocrine purinergic receptor signaling may play a general role in regulating the chemotactic responses of immune cells. Chemotaxis, the movement of cells along chemical gradients, is critical for the recruitment of immune cells to sites of inflammation; however, how cells navigate in chemotactic gradients is poorly understood. Here, we show that macrophages navigate in a gradient of the chemoattractant C5a through the release of adenosine triphosphate (ATP) and autocrine “purinergic feedback loops” that involve receptors for ATP (P2Y2), adenosine diphosphate (ADP) (P2Y12), and adenosine (A2a, A2b, and A3). Whereas macrophages from mice deficient in pannexin-1 (which is part of a putative ATP release pathway), P2Y2, or P2Y12 exhibited efficient chemotactic navigation, chemotaxis was blocked by apyrase, which degrades ATP and ADP, and by the inhibition of multiple purinergic receptors. Furthermore, apyrase impaired the recruitment of monocytes in a mouse model of C5a-induced peritonitis. In addition, we found that stimulation of P2Y2, P2Y12, or adenosine receptors induced the formation of lamellipodial membrane protrusions, causing cell spreading. We propose a model in which autocrine purinergic receptor signaling amplifies and translates chemotactic cues into directional motility.

Flow-induced [Ca2+]i increase depends on nucleotide release and subsequent purinergic signaling in the intact nephron

Flow induces cytosolic Ca(2+) increases ([Ca(2+)](i)) in intact renal tubules, but the mechanism is elusive. Mechanical stimulation in general is known to promote release of nucleotides (ATP/UTP) and trigger auto- and paracrine activation of P2 receptors in renal epithelia. It was hypothesized that the flow-induced [Ca(2+)](i) response in the renal tubule involves mechanically stimulated nucleotide release. This study investigated (1) the expression of P2 receptors in mouse medullary thick ascending limb (mTAL) using P2Y(2) receptor knockout (KO) mice, (2) whether flow increases induce [Ca(2+)](i) elevations in mTAL, and (3) whether this flow response is affected in mice that are deplete of the main purinergic receptor. [Ca(2+)](i) was imaged in perfused mTAL with fura-2 or fluo-4. It is shown that luminal and basolateral P2Y(2) receptors are the main purinergic receptor in this segment. Moreover, the data suggest presence of basolateral P2X receptors. Increases of tubular flow were imposed by promptly rising the inflow pressure, which triggered a marked increase of [Ca(2+)](i). This [Ca(2+)](i) response was significantly reduced in P2Y(2) receptor KO tubules (fura-2 ratio increase WT 0.44 +/- 0.09 [n = 28] versus KO 0.16 +/- 0.04 [n = 13]). Furthermore, the flow response was greatly inhibited with luminal and basolateral scavenging of extracellular ATP (apyrase 7.5 U/ml) or blockage of P2 receptors (suramin 300 microM). The flow response could still be elicited in the absence of extracellular Ca(2+). These results strongly suggest that increase of tubular flow elevates [Ca(2+)](i) in intact renal epithelia. This flow response is caused by release of bilateral nucleotides and subsequent activation of P2 receptors.

α-Hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis

Escherichia coli is the dominant facultative bacterium in the normal intestinal flora. E. coli is, however, also responsible for the majority of serious extraintestinal infections. There are distinct serotypical differences between facultative and invasive E. coli strains. Invasive strains frequently produce virulence factors such as α-hemolysin (HlyA), which causes hemolysis by forming pores in the erythrocyte membrane. The present study reveals that this pore formation triggers purinergic receptor activation to mediate the full hemolytic action. Non-selective ATP-receptor (P2) antagonists (PPADS, suramin) and ATP scavengers (apyrase, hexokinase) concentration dependently inhibited HlyA-induced lysis of equine, murine, and human erythrocytes. The pattern of responsiveness to more selective P2-antagonists implies that both P2X1 and P2X7 receptors are involved in HlyA-induced hemolysis in all three species. In addition, our results also propose a role for the pore protein pannexin1 in HlyA-induced hemolysis, as non-selective inhibitors of this channel significantly reduced hemolysis in the three species. In conclusion, activation of P2X receptors and possibly also pannexins augment hemolysis induced by the bacterial toxin, HlyA. These findings potentially have clinical perspectives as P2 antagonists may ameliorate symptoms during sepsis with hemolytic bacteria.

Distal Colonic K+ Secretion Occurs via BK Channels

K(+) secretion in the kidney and distal colon is a main determinant of K(+) homeostasis. This study investigated the identity of the relevant luminal secretory K(+) ion channel in distal colon. An Ussing chamber was used to measure ion transport in the recently generated BK channel-deficient (BK(-/-)) mice. BK(-/-) mice display a significant colonic epithelial phenotype with (1) lack of Ba(2+)-sensitive resting K(+) secretion, (2) absence of K(+) secretion stimulated by luminal P2Y(2) and P2Y(4) receptors, (3) absence of luminal Ca(2+) ionophore (A23187)-stimulated K(+) secretion, (4) reduced K(+) and increased Na(+) contents in feces, and (5) an increased colonic Na(+) absorption. In contrast, resting and uridine triphosphate (UTP)-stimulated K(+) secretion was not altered in mice that were deficient for the intermediate conductance Ca(2+)-activated K(+) channel SK4. BK channels localize to the luminal membrane of crypt, and reverse transcription-PCR results confirm the expression of the BK channel alpha-subunit in isolated distal colonic crypts. It is concluded that BK channels are the responsible K(+) channels for resting and stimulated Ca(2+)-activated K(+) secretion in mouse distal colon.

Intrarenal Purinergic Signaling in the Control of Renal Tubular Transport

Renal tubular epithelial cells receive hormonal input that regulates volume and electrolyte homeostasis. In addition, numerous intrarenal, local signaling agonists have appeared on the stage of renal physiology. One such system is that of intrarenal purinergic signaling. This system involves all the elements necessary for agonist-mediated intercellular communication. ATP is released from epithelial cells, which activates P2 receptors in the apical and basolateral membrane and thereby modulates tubular transport. Termination of the signal is conducted via the breakdown of ATP to adenosine. Recent far-reaching advances indicate that ATP is often used as a local transmitter for classical sensory transduction. This transmission apparently also applies to sensory functions in the kidney. Locally released ATP is involved in sensing of renal tubular flow or in detecting the distal tubular load of NaCl at the macula densa. This review describes the relevant aspects of local, intrarenal purinergic signaling and outlines its integrative concepts.

K+ secretion activated by luminal P2Y2 and P2Y4 receptors in mouse colon.

Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock‐out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (Vte) (UTP: from −0.81 ± 0.23 to 3.11 ± 0.61 mV, n= 24), an increase of equivalent short circuit current (Isc) by 166.9 ± 22.8 μA cm−2 and a decrease of transepithelial resistance (Rte) from 29.4 ± 2.4 to 23.5 ± 2.0 Ω cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n= 5) and iberiotoxin (240 nm, n= 6), indicating UTP/ATP‐stimulated K+ secretion. RT‐PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP‐stimulated K+ secretion was still present in P2Y2 receptor knock‐out mice, but significantly reduced (ΔVte: 0.83 ± 0.26 mV) compared to wild‐type littermates (ΔVte: 2.08 ± 0.52 mV, n= 9). In P2Y4 receptor knock‐out mice the UTP‐induced K+ secretion was similarly reduced. Luminal UTP‐stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

We have previously shown that a new type of K+ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl− secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K+ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/1, n = 26). Reduction of extracellular Ca2+ to 0.1 mmol/1 (n = 34) led to a reversible reduction of activity of this small channel (SKCa). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K+ channel noise caused by the very small K+ channel increased. Activity of non-selective cation channels (NScat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NScat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca2+ activated and required > 10 μmol/l Ca2+ (cytosolic side = cs). It was reversibly inhibited by ATP (< 1 mmol/1, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SKCa was also Ca2+ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca2+ (cs, n = 12). SKCa was inhibited by Ba2+ (n = 31) and was charybdotoxin sensitive (1 nmol/1) in outside-out basolateral patches (n = 3). Measurements of the Ca2+ activity ([Ca2+]i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K+ concentration to 30 mmol/l, reduced [Ca2+]i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca2+-sensitive K+ channel. This channel is activated promptly by very small increments in [Ca2+]i and is inactivated by a fall in [Ca2+]i induced by forskolin.

P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport

Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl(-) secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K(+) channels. We therefore speculated that a basolateral P2Y(6) receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y(6) receptor using crypt [Ca(2+)](i) measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y(6)elevates cAMP, we coexpressed the P2Y(1) or P2Y(6) receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl(-) currents. In oocytes expressing the P2Y(1) receptor, ATP transiently activated the endogenous Ca(2+)-activated Cl(-) current, but not CFTR. In contrast, in oocytes expressing the P2Y(6)receptor, UDP transiently activated the Ca(2+)-activated Cl(-) current and subsequently CFTR. CFTR Cl(-) currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y(6) receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca(2+)](i) and cAMP. In support of these data P2Y(6) receptor stimulation differentially activates CFTR in Xenopus oocytes.

Basolateral Na+-dependent HCO3− transporter NBCn1-mediated HCO3− influx in rat medullary thick ascending limb

The electroneutral Na+‐dependent HCO3− transporter NBCn1 is strongly expressed in the basolateral membrane of rat medullary thick ascending limb cells (mTAL) and is up‐regulated during NH4+‐induced metabolic acidosis. Here we used in vitro perfusion and BCECF video‐imaging of mTAL tubules to investigate functional localization and regulation of Na+‐dependent HCO3− influx during NH4+‐induced metabolic acidosis. Tubule acidification was induced by removing luminal Na+ (ΔpHi: 0.88 ± 0.11 pH units, n= 10). Subsequently the basolateral perfusion solution was changed to CO2/HCO3− buffer with and without Na+. Basolateral Na+–H+ exchange function was inhibited with amiloride. Na+‐dependent HCO3− influx was determined by calculating initial base flux of Na+‐mediated re‐alkalinization. In untreated animals base flux was 8.4 ± 0.9 pmol min−1 mm−1. A 2.4‐fold increase of base flux to 21.8 ± 3.2 pmol min−1 mm−1 was measured in NH4+‐treated animals (11 days, n= 11). Na+‐dependent re‐alkalinization was significantly larger when compared to control animals (0.38 ± 0.03 versus 0.22 ± 0.02 pH units, n= 10). In addition, Na+‐dependent HCO3− influx was of similar magnitude in chloride‐free medium and also up‐regulated after NH4+ loading. Na+‐dependent HCO3− influx was not inhibited by 400 μm DIDS. A strong up‐regulation of NBCn1 staining was confirmed in immunolabelling experiments. RT‐PCR analysis revealed no evidence for the Na+‐dependent HCO3− transporter NBC4 or the two Na+‐dependent CI−/HCO3− exchangers NCBE and NDCBE. These data strongly indicate that rat mTAL tubules functionally express basolateral DIDS‐insensitive NBCn1. Function and protein are strongly up‐regulated during NH4+‐induced metabolic acidosis. We suggest that NBCn1‐mediated basolateral HCO3− influx is important for basolateral NH3 exit and thus NH4+ excretion by means of setting pHi to a more alkaline value.