Effect of reduced levels of the LDL receptor of Ehrlich ascites tumor cells on cholesterol uptake and cell proliferation: a coculture study with baby hamster kidney cells.

We have introduced a heterologous coculture model between Ehrlich ascites tumor (EAT) and baby hamster kidney cells (PtK2), and we have studied the influence of PtK2 cells and their newly synthesized cholesterol on uptake and tumor cell proliferation. PtK2 cells produce about five times more cholesterol as compared to EAT cells, and they support tumor cell growth in coculture experiments. This growth stimulation is reduced by 80% when EAT cells are cultured in PtK2 cell-derived medium in the presence of a monoclonal anti-low-density lipoprotein receptor (anti-LDL(r)) antibody. Freshly synthesized cholesterol by PtK2 cells is taken up by EAT cells in a time-dependent manner amounting to a threefold increase after 24 h. Alternatively, cholesterol transfer to EAT cells is decreased between 28% and 35%, when PtK2 cell cholesterol synthesis is inhibited in the presence of mevinolin, the specific inhibitor of the hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. Furthermore, lower levels of EAT cell LDL receptor induced by antisense technology reduces cholesterol uptake and cell proliferation. These data demonstrate a metabolic interaction between normal and tumor cells mediated via the exchange of cholesterol, an important membrane constituent.

Tonic and phasic influences of nitric oxide on renal blood flow autoregulation in conscious dogs.

The aim of this study was to investigate the influence of the mean level and phasic modulation of NO on the dynamic autoregulation of renal blood flow (RBF). Transfer functions were calculated from spontaneous fluctuations of RBF and arterial pressure (AP) in conscious resting dogs for 2 h under control conditions, after NO synthase (NOS) inhibition [NG-nitro-L-arginine methyl ester hydrochloride (L-NAME)] and after L-NAME followed by a continuous infusion of an NO donor [S-nitroso-N-acetyl-DL-penicillamine (SNAP)]. After L-NAME (n = 7) AP was elevated, heart rate (HR) and RBF were reduced. The gain of the transfer function above 0.08 Hz was increased, compatible with enhanced resonance of the myogenic response. A peak of high gain around 0.03 Hz, reflecting oscillations of the tubuloglomerular feedback (TGF), was not affected. The gain below 0.01 Hz, was elevated, but still less than 0 dB, indicating diminished but not abolished autoregulation. After L-NAME and SNAP (n = 5), mean AP and RBF were not changed, but HR was slightly elevated. The gain above 0.08 Hz and the peak of high gain at 0.03 Hz were not affected. The gain below 0.01 Hz was elevated, but smaller than 0 dB. It is concluded that NO may help to prevent resonance of the myogenic response depending on the mean level of NO. The feedback oscillations of the TGF are not affected by NO. NO contributes to the autoregulation below 0.01 Hz due to phasic modulation independent of its mean level.

Cholesterol-induced growth stimulation, macromolecule synthesis, and increased phosphoinositide metabolism of ascites tumour cells in culture.

Ascites tumour cells have previously been shown by us to require exogenous cholesterol for growth. To investigate further this phenomenon, we have used, in addition to free cholesterol, cholesterol complexed to digitonine, to elaborate the specificity of this growth-controlling process using a chemically defined medium. Our data show that only free cholesterol stimulates cell growth and macromolecule synthesis in a dose-dependent manner, suggesting that the proper embedding of the sterol into the membrane is a prerequisite for its function. Furthermore, studies have been performed on the influence of cholesterol on the phosphoinositide metabolism of our cells, as phosphoinositides furnish important second messenger molecules in the cascade of signal transduction. We could show that cholesterol stimulates a transient release of inositol trisphosphate and other inositol phosphates by inducing the activation of phospholipase C (PLC). PLC activation by a factor of about 3 with phosphatidylinositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate as substrates could be measured directly by using a partially purified membrane preparation. This enzyme activity was found to be strongly dependent on free Ca2+ ions with optimal concentrations of 100 nM for cholesterol- and 50 nM for cholesterol-digitonide-treated cells. Ca2+ concentration for half-maximum activation, however, was identical under both conditions. Phospholipase C activity could be synergistically increased about 2-fold with 25 microg GTP gammaS in cholesterol-digitonide-treated cells as well, suggesting that the coupling between phospholipase C and the G-protein was not disturbed by the complex. These data demonstrate a functional role of cholesterol on cell growth, macromolecule synthesis, and phosphoinositide metabolism mediating the release of important second messenger molecules.

Autoregulation of renal blood flow in the conscious dog and the contribution of the tubuloglomerular feedback.

1. The aim of this study was to investigate the autoregulation of renal blood flow under physiological conditions, when challenged by the normal pressure fluctuations, and the contribution of the tubuloglomerular feedback (TGF). 2. The transfer function between 0.0018 and 0.5 Hz was calculated from the spontaneous fluctuations in renal arterial blood pressure (RABP) and renal blood flow (RBF) in conscious resting dogs. The response of RBF to stepwise artificially induced reductions in RABP was also studied (stepwise autoregulation). 3. Under control conditions (n = 12 dogs), the gain of the transfer function started to decrease, indicating improving autoregulation, below 0.06-0.15 Hz (t = 7-17 s). At 0.027 Hz a prominent peak of high gain was found. Below 0.01 Hz (t > 100 s), the gain reached a minimum (maximal autoregulation) of -6.3 +/- 0.6 dB. The stepwise autoregulation (n = 4) was much stronger (-19.5 dB). The time delay of the transfer function was remarkably constant from 0.03 to 0.08 Hz (high frequency (HF) range) at 1.7s and from 0.0034 to 0.01 Hz (low frequency) (LF) range) at 14.3 s, respectively. 4. Nifedipine, infused into the renal artery, abolished the stepwise autoregulation (-2.0 +/- 1.1 dB, n = 3). The gain of the transfer function (n = 4) remained high down to 0.0034 Hz; in the LF range it was higher than in the control (0.3 +/- 1.0 dB, P < 0.05). The time delay in the HF range was reduced to 0.5 s (P < 0.05). 5. After ganglionic blockade (n = 7) no major changes in the transfer function were observed. 6. Under furosemide (frusemide) (40 mg + 10 MG h-1 or 300 mg + 300 mg h-1 i.v..) the stepwise autoregulation was impaired to -7.8 +/- 0.3 or 6.7 +/- 1.9 dB, respectively (n = 4). In the transfer function (n = 7 or n = 4) the peak at 0.027 Hz was abolished. The delay in the LF range was reduced to -1.1 or -1.6 s, respectively. The transfer gain in the LF range (-5.5 +/- 1.2 or -3.8 +/- 0.8 dB, respectively) did not differ from the control but was smaller than that under nifedipine (P < 0.05). 7. It is concluded that the ample capacity for regulation of RBF is only partially employed under physiological conditions. The abolition by nifedipine and the negligible effect of ganglionic blockade show that above 0.0034 Hz it is almost exclusively due to autoregulation by the kidney itself. TGF contributes to the maximum autoregulatory capacity, but it is not required for the level of autoregulation expended under physiological conditions. Around 0.027 Hz, TGF even reduces the degree of autoregulation.

Frequency domain of renal autoregulation in the conscious dog.

The dynamic range in which renal blood flow (RBF) autoregulation occurs was determined in eight conscious foxhounds chronically catheterized in the abdominal aorta and implanted with a transit-time flow probe over the renal artery. Sinusoidal driving pressures (amplitude of 10 mmHg) were forced on the renal arterial pressure at different frequencies by a servo-control device, and transfer functions were calculated. Only one frequency range was found below which the gain of the transfer function declined and in which the phase angle increased (n = 8). This indicates the presence of a potent mechanism for renal autoregulation in the examined frequency range between 0.0031 and 0.08 Hz, which buffers changes in blood flow < 0.02 Hz. After furosemide treatment, one indicator for autoregulation (phase shift of transfer function) was significantly blunted at low frequencies (n = 6). Furosemide, however, did not reduce the phase shift to zero, suggesting that some autoregulation still remained in the frequency range between 0.04 and 0.08 Hz. In conclusion, autoregulation of RBF during sinusoidal changes in driving pressure between 0.0031 and 0.02 Hz is mediated by a single mechanism, which can be blocked by the acute administration of furosemide. The residual phase shift between arterial pressure and RBF in the transfer function observed during sinusoidal changes in driving pressure between 0.04 and 0.08 Hz suggests the presence of a second mechanism for RBF autoregulation.

The blood pressure buffering capacity of nitric oxide by comparison to the baroreceptor reflex.

To compare the contribution of nitric oxide (NO) to the buffering of short-term and circadian fluctuations of arterial blood pressure with that of the baroreceptor reflex, conscious foxhounds were subjected to continuous 24-h blood pressure recordings. A pressure transducer was placed into the lumen of the abdominal aorta. Telemetry recordings were done under control conditions, following blockade of NO formation by intravenous bolus injection of NG-nitro-L-arginine (L-NNA; 16.5 +/- 2 mg/kg body wt) and after total sinoaortic and cardiopulmonary denervation in five dogs each. L-NNA produced a sustained elevation of mean arterial pressure (MAP; 137.2 +/- 6.4 mmHg vs. control, 112.9 +/- 3.7 mmHg). After denervation, no significant increase of MAP was found (113.5 +/- 4.1 mmHg), but the standard deviation of the MAP histogram was significantly greater (22.5 +/- 3.1 vs. 10.6 +/- 0.9 mmHg, P < 0.05). Sequential spectral analysis showed that total power between 0 and 0.5 Hz was elevated more than twofold after L-NNA (P < 0.05). This was due primarily to increased power in the range above 0.1 Hz. After denervation, total power increased about three-fold (P < 0.05), almost exclusively occurring below 0.04 Hz. Power in the range above 0.2 Hz was diminished, although not significantly. It is concluded that in the conscious dog, NO, as well as the baroreceptor reflex, is an effective blood pressure buffer. NO is most effective above 0.1 Hz, whereas the baroreceptors primarily buffer fluctuations slower than 0.04 Hz.

Free cholesterol induces activation but not translocation of protein kinase C in cultured ascites tumour cells.

Ascites tumour cells, cultivated in a serum replacement medium with low mitogenic activity supplemented with free cholesterol or cholesterol complexed to digitonine, were used to study protein kinase C (PKC) activation and its translocation from the cytosol to the membrane compartment. Activity of partially purified PKC was assessed by the Ca2+/phospholipid-dependent phosphorylation of histone as substrate. Staurosporine-sensitive PKC activity was increased by a factor of about three in the membrane compartment as the result of cholesterol supplementation with about 90% of total activity in the membrane, and the remaining 10% in the cytosolic fraction. A translocation of enzyme mass from the cytosol to the membrane compartment was not induced under these conditions, as documented by the enzyme-linked immuno adsorbent assay using a monoclonal anti-PKC antibody. In cells cultivated in the presence of the cholesterol-digitonide complex PKC activation was not observed, suggesting an inadequate utilization of cholesterol as membrane constituent.

Spectrum analysis of sympathetic nerve activity and blood pressure in conscious rats.

This study tests whether the power spectrum of blood pressure (BP) provides information toward the sympathovagal balance of BP control by comparing the BP (femoral arterial catheter) spectrum with the spectrum of the efferent sympathetic nerve activity (SNA, bipolar electrode around splanchnic nerve). A remarkable resemblance between both spectra was found. A high-frequency component (HF) linked to respiration and a slower fluctuation type between 0.15 and 0.6 Hz (LF) were identified. There was a large and significant coherence only in the HF range of the BP and SNA power spectrum (P < 0.01). The phase lag of SNA and BP was roughly 200 ms. The recordings were repeated during pharmacological blockade in nine Wistar-Kyoto rats (WKY) and nine spontaneously hypertensive rats (SHR). alpha 1-Adrenoceptor blockade (prazosin) reduced the proportional LF power of BP in both rat strains (WKY P < 0.01, SHR P < 0.05) in favor of HF (WKY P < 0.01, SHR P < 0.01). Parasympathetic blockade (methylscopolamine) had no effect on proportions of power. Similarly, there were no significant differences in the proportional HF and LF power spectra of WKY and SHR. These data provide direct evidence for a relationship between the BP and SNA power spectra; however, only the acute changes in the sympathetic tone changed the LF-HF relationship.

Ca2+ and partly GTP gamma S-dependent particulate phospholipase C hydrolyzing phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is inhibited by diacyl(acyl-acetyl) glycerols.

The activity of a phosphodiesterase of the phospholipase C (PLC) type and factors influencing its activity were studied in ascites tumor cells. The enzyme confined to the 12,000 x g particulate fraction hydrolyses inositol phospholipids, with preference for phosphatidylinositol 4-phosphate (PtdIns(4)P) over phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), exhibiting maximum values of 61 and 15 nmol/min per mg protein, respectively, at a pH optimum of 5.5. The phosphodiesterase, which is strongly Ca2+ dependent with optimal free Ca2+ concentrations between 20 and 100 nM for both substrates, is almost completely inhibited (93-95%) in the presence of 2 mM EGTA. Only the PLC acting on PtdIns(4,5)P2 is significantly activated in the presence of 6-60 microM GTP gamma S. The low extent of enzymatic activity in the presence of 5 mM MgCl2 or chelating agents is suggestive of inositolphosphatase activity which is supported by the determination of small amounts of myo-inositol during HPLC analyses. Both dioleoylglycerol (DAG) and the membrane-permeable 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibit PLC activity, exhibiting IC50 values of 5 microM with PtdIns(4)P and approx. 10 microM with PtdIns(4,5)P2 as substrate and maximum inhibition up to 60% (DAG) and 80% (OAG). These data are indicative of a mechanism of direct negative feedback regulation of the enzyme by diglycerides which may explain the observed long-term effects of OAG on PLC activity in cell culture experiments.

Phasic and 24-h blood pressure control by endothelium-derived relaxing factor in conscious dogs.

The effects of blocking endothelium-derived relaxing factor (EDRF) on 24-h blood pressure and heart rate (HR) were examined in six conscious freely moving foxhounds. The hypothesis tested was that shear stress-dependent EDRF release acts as a physiological blood pressure buffer. Telemetry recordings were obtained before and after the administration of the false substrate for EDRF synthesis NG-nitro-L-arginine (L-NNA, 16.5 +/- 2 mg/kg body wt iv). In response to L-NNA, mean arterial blood pressure (MAP) increased from 116 +/- 5 to 134 +/- 5 mmHg (P less than 0.01) and HR decreased from 97 +/- 6 to 68 +/- 3 beats/min over the entire 24-h period (P less than 0.01). The overall variability of MAP (as indicated by SD of frequency distribution) increased modestly from 9.5 +/- 0.4 to 11.7 +/- 1.1 mmHg (P less than 0.05). A sequential spectral analysis of blood pressure showed a 2.1-fold increase of power in the frequency range of 0.01-0.5 Hz (P less than 0.05) after L-NNA was given. In conclusion, blockade of EDRF led to a sustained hypertension throughout the whole 24-h recordings. Furthermore, EDRF acted as a physiological blood pressure buffer in the frequency range below 0.5 Hz.