Hyperosmotic and isosmotic shrinkage differentially affect protein phosphorylation and ion transport.

In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%-50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na(+),K(+)-ATPase and Na(+),P(i) cotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na(+),K(+)-ATPase and did not change Na(+),P(i) cotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ∼7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.

Long-term normalization of blood pressure in SHR and 1-kidney 1-clip rats by synthetic precursor of stable PAF analogue without systemic effects in normotensive rats.

This study characterized the actions of the newly synthesized PAF precursor 1-hexadecyl-2-alkylcarbamoyl-glycerol (HAG) on blood pressure (BP) in male spontaneously hypertensive rats (SHR), SHR-stroke prone (SHRSP) and Wistar rats with 1-kidney 1-clip (1K1C) renovascular hypertension used as experimental models of human primary and secondary hypertension. Systolic blood pressure (SBP) in the tail artery and mean arterial pressure (MAP) in the abdominal aorta were measured by tail plethysmography and invasive pressure transducer, respectively. Intravenous treatment with 1mg/kg HAG in SHR resulted in a rapid decline of MAP from 151±4 to 127±4mmHg in 50min (p<0.001) that was maintained for 24h after injection (128±5mmHg, p<0.01). We also observed a profound hypotensive effect of HAG in SHRSP but not in normotensive Wistar rats. In 1K1C rats, the magnitude of the BP decline evoked by HAG was correlated with MAP measured before drug administration (R=0.74, p<0.005). In 1K1C rats with SBP>140mmHg, 5mg/kg/48h HAG, given orally for 14 days, decreased SBP by 20-30mmHg without an increase in the death rate and other adverse effects. Thus, our results show that intravenous and oral administration of HAG led to a long-lasting reduction of BP in experimental models of primary and secondary hypertension. In contrast to PAF and its derivatives, the hypotensive action of HAG was preserved for 24h after a single administration, was absent in normotensive animals, and was not accompanied by visible side-effects, at least during 2 weeks of treatment.

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na(+), K (+), 2Cl (-) cotransport-dependent signaling.

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog alpha,beta-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X(1) receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y(6) receptors. Inhibition of Na(+), K(+), 2Cl(-) cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y(6)-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y(6)-mediated signaling should be examined further.

Excitation-contraction coupling in resistance mesenteric arteries: evidence for NKCC1-mediated pathway.

Bumetanide and other high-ceiling diuretics (HCD) attenuate myogenic tone and contractions of vascular smooth muscle cells (VSMC) triggered by diverse stimuli. HCD outcome may be mediated by their interaction with NKCC1, the only isoform of Na(+), K(+), 2Cl(-) cotransporter expressed in VSMC as well as with targets distinct from this carrier. To examine these hypotheses, we compared the effect of bumetanide on contractions of mesenteric arteries from wild-type and NKCC1 knockout mice. In mesenteric arteries from wild-type controls, 100 microM bumetanide evoked a decrease of up to 4-fold in myogenic tone and contractions triggered by modest [K(+)](o)-induced depolarization, phenylephrine and UTP. These actions of bumetanide were preserved after inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester, but were absent in mesenteric arteries from NKCC1(-/-) mice. The data show that bumetanide inhibits VSMC contractile responses via its interaction with NKCC1 and independently of nitric oxide production by endothelial cells.

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence.

The mechanisms of cell death signaling triggered by cardiotonic steroids are poorly understood. Based on massive detachment of ouabain-treated Madin-Darby canine kidney (MDCK) cells, it may be proposed that the cytotoxic action of these compounds is mediated by anoikis, i.e. a particular mode of death occurring in cells lacking cell-to-extracellular matrix interactions. We tested this hypothesis. Six hour incubation of MDCK cells with ouabain, marinobufagenin or K+-free medium almost completely blocked Na+,K+-ATPase, increased Na (i) + content by approximately 10-fold and suppressed cell attachment to regular-plastic-plates by up to 5-fold. In contrast, the death of attached cells was observed after 24-h incubation with ouabain but not in the presence of marinobufagenin or K+-free medium. Cells treated with ouabain and undergoing anoikis on ultra-low attachment plates exhibited different cell volume behaviour, i.e. swelling and shrinkage, respectively. The pan-caspase inhibitor z-VAD.fmk and the protein kinase C activator PMA rescued MDCK cells from anoikis but did not influence the survival of ouabain-treated cells, whereas medium acidification from pH 7.2 to 6.7 almost completely abolished the cytotoxic action of ouabain, but did not significantly affect anoikis. Our results show that the Na (i) + ,K (i) + -independent mode of MDCK cell death evoked by ouabain is not mediated by anoikis.

Extracellular calcium is required for the maintenance of plasma membrane integrity in nucleated cells.

In contrast to rat and human erythrocytes, nucleated erythrocytes from two fish species (Cyprinus carpio and Salmo trutta) underwent almost complete haemolysis in 20 min of EDTA addition. Using Ca2+/Mg2+ EGTA-citrate buffer, we observed that half-maximal haemolysis of fish erythrocytes occurs at [Ca2+]o approximately 10 microM independently of extracellular Mg2+ concentration. Attenuation of [Ca2+]o with EGTA also decreased stability of the plasma membrane of vascular smooth muscle cells (VSMC) and HeLa cells, indicated by a three- to five-fold elevation of lactate dehydrogenase release and passive permeability of plasma membrane for Na+. In VSMC, EGTA lowered [Ca2+]i by approximately 20%. This effect was absent in VSMC-loaded with the intracellular Ca2+ chelator BAPTA. In contrast to EGTA, BAPTA did not affect haemoglobin release from fish erythrocytes and passive permeability for Na+ in VSMC. Viewed collectively, our data show that in nucleated cells, extracellular Ca2+ plays a crucial role in the maintenance of plasma membrane integrity.

Do we know the absolute values of intracellular free calcium concentration?

More than 20 years ago, it was shown that the addition of EGTA increases the affinity of the plasma membrane Ca2+ pump for Ca2+ by an order of magnitude. The left-hand shift of Ca2+-dependencies in the presence of EGTA has been also documented in studies of the sarcoplasmic reticulum Ca2+ pump, mitochondrial Ca2+-transporter as well as Ca2+-binding by calmodulin and troponin C. These data allow us to hypothesise that this effect is caused by an admixture of di- and trivalent cations possessing high affinity for EGTA and interacting with Ca2+-transporting and binding proteins. Here, we propose that polyvalent cations affect the estimation of absolute values of free intracellular Ca2+ concentration. Indeed, EGTA sharply increases the apparent affinity of the fluorescent Ca2+ indicators quin-2 and fluo-3 for Ca2+. The impact of polyvalent cations on Ca2+ measurement was further confirmed by the study showing the high sensitivity of Ca2+-induced fluo-3 fluorescence to Mn2+, Fe2+, Cu2+, and Co2+ seen in the absence of EGTA.