Ca2+-transients induced by K+ channel openers in isolated coronary capillaries.

To study the role of endothelial ATP-sensitive K+ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca2+]i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca2+]i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K+ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca2+]i followed by a sustained elevation of [Ca2+]i (19 of 40 experiments). [Ca2+]i in the endothelial cells increased from 32 +/- 7 nM at rest to 66 +/- 11 nM at the peak (n = 19). One third of the [Ca2+]i-transients showed irregular oscillations of [Ca2+]i. No significant difference in the [Ca2+]i-response induced by 100 nM or 1 muM diazoxide was found. Similar results were obtained with the K+ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca2+]i with fluorometric methods indicated that the hyperpolarization but not the [Ca2+]i-transient could be repeatedly induced in a single capillary by the K+ channel openers. Electrophysiological recordings of the membrane potential using the "perforated patch" method (n = 4), showed that rilmakalim (1 muM) induced hyperpolarization of capillaries towards the K+ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K+ channel openers induce [Ca2+]i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of KATP-channel in coronary capillaries characterised by high sensitivity to diazoxide.

Endothelial ATP-sensitive potassium channels: a new therapeutic target in cardiovascular medicine?

Recent electrophysiological studies have demonstrated convincingly that ATP-sensitive potassium channels (KATP) are expressed in macro- and microvascular endothelial cells from heart and brain. Several reports have indirectly demonstrated the contribution of endothelial KATP to the regulation of vascular tone, eg, during hypoxia. Experimentally, endothelial KATP can be activated by energy depletion of cells or by adding synthetic potassium channel openers (PCOs). In endothelial cells, in contrast to vascular smooth muscle cells (VSMC), hyperpolarization induced by KATP opening can result in an increase of [Ca2+]i and, further, to enhanced NO and prostanoid synthesis. Although this mechanism is highly speculative, synthetic PCOs, directed against endothelial KATP, may counteract atherogenic stimuli (hypercholesterolemia and hypertension) and/or even suppress symptoms occurring in atherosclerosis (proliferation of VSMC, thrombogenesis). Further-more, the cardioprotective effects of PCOs may be mediated, at least in part, by an activation of endothelial KATP.

Hyperpolarization of isolated capillaries from guinea-pig heart induced by K+ channel openers and glucose deprivation.

1. The present study was designed to test if microvascular coronary endothelial cells express ATP-sensitive K+ channels (KATP channels). We performed microfluorometric measurements of the membrane potential of freshly isolated guinea-pig coronary capillaries equilibrated with the voltage-sensitive dye bis-oxonol (bis-[1,3-dibutylbarbituric acid] trimethineoxonol, [DiBAC4(3)]). 2. The resting membrane potential of capillaries in physiological salt solution was -46 +/- 4.2 mV (n = 8) at room temperature (22 degrees C) as determined after calibration of the fluorescence using the Na(+)-K+ ionophore gramicidin in the presence of different K+ concentrations. Spontaneous membrane potential fluctuations of 10-20 mV amplitude were often observed. 3. A reversible, sustained hyperpolarization to a new membrane potential close to the K+ equilibrium potential (EK) could be induced by application of the K+ channel openers HOE 234 (100 nM to 1 microM), diazoxide (10 PM to 100 nM) or pinacidil (100 nM). Subsequent addition of glibenclamide (200 nM to 2 microM) reversed this hyperpolarization. 4. A glibenclamide-sensitive hyperpolarization of coronary capillaries to values near EK was also observed upon omission of D-glucose (10 mM) from the superfusing solution or by substituting L-glucose for D-glucose. Maximum hyperpolarization was reached in less than 10 min. 5. Our results suggest that microvascular coronary endothelial cells express KATP channels which may be activated during hypoglycaemia.

Plasma membrane-associated aminopeptidase activities in Chlamydomonas reinhardtii and their biochemical characterization.

High aminopeptidase (Apase) activities were found on intact unicellular algae cells. Several lines of evidence strongly indicate that the external Apases on Chlamydomonas reinhardtii (a green alga) cells, characterized in the present study, are plasma membrane-associated proteinases and not secreted in the cell wall or the surrounding medium. This is shown by enzyme activities also detected on a cell wall deficient mutant of C. reinhardtii and by the finding that in assay media and algal conditioned nutrient solutions, respectively, no Apase activities were found after removal of cells. In C. reinhardtii at least two in vivo Apases, one L-leucine-p-nitroanilide and one L-alanine-p-nitroanilide hydrolyzing enzyme (in vivo LeuNAase and AlaNAase, respectively) as well as one in vivo endoproteinase, capable of cleaving carboxybenzoylleucine-p-nitroanilide (CBZLeuNAase), were clearly distinguished by their pH optima for activity and characteristics towards various chemical compounds. In vivo LeuNAase, which cannot unequivocally classified as a metallo- or serine-type proteinase, showed optimum activities between pH 7 and 8.5, stimulation of activity by 1,10-phenanthroline (161%), 2-fold higher activity with L-phenylalanine-p-nitroanilide than with LeuNA and a Km value of 40 microM LeuNA. In vivo AlaNAase favored alkaline pH values, had a Km value of 1.45 mM AlaNA and is probably a metallopeptidase as indicated by 2-fold enhancement of enzyme activity by 5 microM Co2+ and strong inhibition with 1,10-phenanthroline. This enzyme was inhibited completely by a 30 min incubation with 10 microM Hg2+ at room temperature, indicating sensitive SH-groups. In contrast, activity was stimulated 205% by 20 mM iodoacetate in the assay buffer. Both in vivo Apases were efficiently inhibited by 10 mM Pefabloc SC, a serine-type proteinase inhibitor and by two compounds, not yet described as proteinase inhibitors: methyljasmonate, a plant hormone, and dibucaine, a local anestheticum. The latter compound showed the most powerful inhibition on in vivo and in vitro LeuNAase of all reagents tested. From the distribution of Apase activities and characteristics in the cell, it is hypothesized that at least the LeuNAase dissociates easily from the plasma membrane during preparation of cell extracts and binds then unspecifically to various membrane fractions. In conclusion, this is the first report on the existence of external Apase activities on plant cells providing an easy-to-perform, rapid and reliable assay method for these enzymes.

Influence of a high Mn supply on Norway spruce (Picea abies (L.) Karst.) seedlings in relation to the nitrogen source.

Effects of 3, 25, 100, 200 and 800 microM Mn on biomass and pigment, starch and nitrate concentrations were studied in Norway spruce (Picea abies (L.) Karst.) seedlings grown with either NO(3) (-) or NH(4) (+) as the sole nitrogen source. After 77 days of exposure to 800 microM Mn, shoot growth had ceased in about 50% of the seedlings independently of the N source. Despite high Mn concentrations in roots and shoots of the Mn-treated seedlings, no visible symptoms of Mn toxicity were evident. The rate of root elongation was decreased by treatment with >/= 200 microM Mn when N was supplied as NO(3) (-), but not when it was supplied as NH(4) (+). This difference could be attributed to the higher Mn concentrations in root tips of the NO(3) (-)-grown seedlings compared with the NH(4) (+)-grown seedlings. In Mn-treated seedlings, the concentration of Mg, and to a lesser extent that of Ca, decreased. Depletion of these elements might account for the observed growth depression. Potassium concentrations were similar in the control and Mn-treated seedlings. Treatment of seedlings with 800 microM Mn for 50 days led to several physiological changes: starch accumulated, the concentrations of nitrate and phenolic compounds increased, pigment concentrations decreased, and in vivo nitrate reductase activity in roots was reduced.

Vegetative storage proteins in poplar : induction and characterization of a 32- and a 36-kilodalton polypeptide.

Bark, wood, and root tissues of several Populus species contain a 32- and a 36-kilodalton polypeptide which undergo seasonal fluctuations and are considered to be storage proteins. These two proteins are abundant in winter and not detectable in summer as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection. An antibody raised against the 32-kilodalton storage protein of Populus trichocarpa (T. & G.) cross-reacts with the 36-kilodalton protein of this species. The synthesis of the 32- and 36-kilodalton proteins can be induced in micropropagated plants by short-day conditions in the growth chamber. These proteins are highly abundant in structural roots, bark, and wood and combined represent >25% of the total soluble proteins in these tissues. Nitrate concentration in the leaves and nitrate uptake rate decreased dramatically when LD plants were transferred to short-day conditions; the protein content in leaves was unaffected. A decrease of the 32- and 36-kilodalton polypeptides occurs after transferring induced plants back to LD conditions. Both polypeptides are glycosylated and can be efficiently purified by affinity chromatography using concanavalin A-Sepharose 4B. The 32- and the 36-kilodalton polypeptides have identical basic isoelectric points and both consist of at least three isoforms. The storage proteins show a loss in apparent molecular mass after deglycosylation with trifluoromethanesulfonic acid. It is concluded that the 32- and 36-kilodalton polypeptides are glycoforms differing only in the extent of glycosylation. The relative molecular mass of the native storage protein was estimated to be 58 kilodalton, using gel filtration. From the molecular mass and the elution pattern it is supposed that the storage protein occurs as a heterodimer composed of one 32- and one 36-kilodalton subunit. Preliminary data suggest the involvement of the phytochrome system in the induction process of the 32- and 36-kilodalton polypeptides.