The hypothesis that cell primary cilium is solely responsible for the flow-induced Ca2+ response in MDCK cells was tested by removal of the cilia from mature, responsive cells. Incubation of the cells with 4 mM chloral hydrate for 68 hours resulted in the complete loss of the primary cilia and in disorganization of microtubules, as visualized by immunofluorescence. When intracellular Ca2+ concentration was measured with Fluo-4, the elevation that normally accompanies an increase in fluid flow was abolished after 20 hours exposure to chloral hydrate. At this time, the primary cilia still remained attached to the cells but had become twisted and flexible. Twenty-four hours after return of the deciliated cells to normal medium, intracellular microtubule organization appeared normal, but primary cilia had not yet been expressed. The cells failed to increase intracellular Ca2+ in response to fluid flow until after they had been in normal medium for 120 hours, at which time the primary cilia were 3-4 microm long. Chloral hydrate did not impair the Ca2+ mobilization machinery, as the Ca2+ response to mechanical contact and the spread to neighboring cells was unaffected by the drug. We conclude that the primary cilium is the only sensor for the flow-induced Ca2+ response in MDCK cells and estimate that a single mechanically sensitive channel in the cilium could provide the requisite Ca2+ influx.

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 nonexcitable 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.

The primary cilium, an organelle largely ignored by physiologists, functions both as a mechano-sensor and a chemo-sensor in renal tubular epithelia. This forgotten structure is critically involved in the determination of left-right sidedness during development and is a key factor in the development of polycystic kidney disease, as well as a number of other abnormalities. This review provides an update of our current understanding about the function of primary cilia. Much new information obtained in the past five years has been stimulated, in part, by discoveries of the primary cilium's key role in the genesis of polycystic kidney disease as well as its involvement in determination of left-right axis asymmetry. Here we focus on the various functions of the primary cilium rather than on its role in pathology.

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 ␣-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 P2X 1 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.alpha-hemolysin ͉ E. coli ͉ erythrocytes ͉ hemolysis ͉ P2X

The primary cilium is mechanically sensitive and serves as a flow sensor in cultured renal epithelia. Bending the cilium by mechanical means or flow causes a large, prolonged transient increase in intracellular calcium. The mechanically sensitive protein in the cilium is a polycystin.

Flow induces cytosolic Ca 2ϩ increases ([Ca 2ϩ ] 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 flowinduced [Ca 2ϩ ] 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 2ϩ ] i elevations in mTAL, and (3) whether this flow response is affected in mice that are deplete of the main purinergic receptor. [Ca 2ϩ ] 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 2ϩ ] i . This [Ca 2ϩ ] i response was significantly reduced in P2Y 2 receptor KO tubules (fura-2 ratio increase WT 0.44 Ϯ 0.09 [n ϭ 28] versus KO 0.16 Ϯ 0.04 [n ϭ 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 M). The flow response could still be elicited in the absence of extracellular Ca 2ϩ . These results strongly suggest that increase of tubular flow elevates [Ca 2ϩ ] i in intact renal epithelia. This flow response is caused by release of bilateral nucleotides and subsequent activation of P2 receptors.

Reducing luminal NaCl concentration in the macula densa region of the nephron stimulates renin secretion, and this response is blocked by a specific inhibitor of cyclooxygenase-2 (COX-2) (Traynor, T. R., Smart, A., Briggs, J. P., and Schnermann, J. (1999) Am. J. Physiol. Renal Physiol. 277, F706 -710). To study whether low NaCl activates COX-2 activity or expression we clonally derived a macula densa cell line (MMDD1 cells) from SV-40 transgenic mice using fluorescence-activated cell sorting of renal tubular cells labeled with segment-specific fluorescent lectins. MMDD1 cells express COX-2, bNOS, NKCC2, and ROMK, but not Tamm-Horsfall protein, and showed rapid 86 Rb ؉ uptake that was inhibited by a reduction in NaCl concentration and by bumetanide or furosemide. Isosmotic exposure of MMDD1 cells to low NaCl (60 mM) caused a prompt and time-dependent stimulation of prostaglandin E 2 (PGE 2 ) release that was prevented by the COX-2 specific inhibitor NS-398 (10 M). Reducing NaCl to 60 and 6 mM for 16 h increased COX-2 expression in a chloride-dependent fashion. Low NaCl phosphorylated p38 kinase within 30 min and ERK1/2 kinases within 15 min without changing total MAP kinase levels. Low NaCl-stimulated PGE 2 release and COX-2 expression was inhibited by SB 203580 and PD 98059 (10 M), inhibitors of p38 and ERK kinase pathways. We conclude that low chloride stimulates PGE 2 release and COX-2 expression in MMDD1 cells through activation of MAP kinases.

Calcium (Ca) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca-ATPase (SERCA), disrupts Ca homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 μm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca stores. This Ca depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca levels. Moreover, knockdown of two key store-operated Ca entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca levels and SOCE are not required.