Preservation of Adrenoceptor and Endothelin Receptor Mediated Vasoconstriction and of Endothelium-Dependent Relaxation after Cold Storage of Explanted Blood Vessels for ex vivo Analyses.

INTRODUCTION: Adrenoceptor and endothelin (ET) receptor-mediated vasoconstriction as well as endothelium-dependent vasodilation of human saphenous veins were compared before and after 20 h of cold storage. METHODS: Contractile responses to potassium chloride (KCl), norepinephrine (NE), and ET-1 as well as vasodilator responses to acetylcholine (ACh) were evaluated. RESULTS: Storage in HEPES-supplemented Dulbecco's modified Eagle's medium (HDMEM) diminished KCl induced contractile forces to 71% (p = 0.002) and NE induced contractions to 80% (p = 0.037), in contrast to HEPES-supplemented Krebs-Henseleit solution (HKH) and TiProtec solution. KCl-normalized NE contractions were not affected by storage. NE EC50 values were slightly lower (7.1E-8 vs. 7.5E-8, p = 0.019) after storage in HKH, with no changes after storage in the other solutions. Endothelium-dependent responses to ACh were not affected by storage. ET-1 induced contractions were attenuated after storage in HDMEM (77%, p = 0.002), HKH (75%, p = 0.020), and TiProtec (73%, p = 0.010) with no changes in normalized constrictions. ET-1 EC50 values were not affected by storage. CONCLUSION: Loss of contractility after storage in HDMEM may reflect the lower content of dextrose. There was no specific attenuation of adrenoceptor, ET-receptor, or ACh receptor mediated signal transduction after storage in any of the media. HKH or TiProtec are equally suitable cold storage solutions for ex vivo measurements.

Pectic acid effects on prolactin secretion in GH3/B6 rat pituitary cell line.

BACKGROUND: Pectic acid extracted from plants increases the secretion of prolactin (PRL) when injected intravenously into ewes or fed to rats. Fragments of ewe hypophysis and lactating rabbit mammary gland incubated in vitro in the presence of pectic acid secreted more PRL and caseins compared to the controls. However, it is not known whether pectic acid directly stimulates PRL secretion in pituitary or interference of factors from hypophysis is required for this process. METHODS: GH3/B6 cells, a clonal strain of rat pituitary, were cultured and incubated with pectic acid (2.5-100 microg/mL). The integrity of cells was examined under pectic acid treatment microscopically. Controls or pectic acid treated cells were assayed for their ability to produce PRL. The PRL was assayed by Western-blotting and Radioimmunoassay. RESULTS: pectic acid did not have any significant effect on the viability of cells. After being incubated with pectic acid, the cells started to become circular and protuberant shape. The maximum stimulation and PRL secretion occurred at 100 microg/mL concentration within 30 min of incubation with pectic acid. CONCLUSION: pectic acid could stimulate the release of PRL in GH3/B6 cells in the short-term incubation. This result suggested that pectic acid is a non-toxic agent that could directly stimulate PRL secretion in pituitary cells without any interference of hypophysis.

Feedback from horizontal cells to rod photoreceptors in vertebrate retina.

Retinal horizontal cells (HCs) provide negative feedback to cones, but, largely because annular illumination fails to evoke a depolarizing response in rods, it is widely believed that there is no feedback from HCs to rods. However, feedback from HCs to cones involves small changes in the calcium current (I(Ca)) that do not always generate detectable depolarizing responses. We therefore recorded I(Ca) directly from rods to test whether they were modulated by feedback from HCs. To circumvent problems presented by overlapping receptive fields of HCs and rods, we manipulated the membrane potential of voltage-clamped HCs while simultaneously recording from rods in a salamander retinal slice preparation. Like HC feedback in cones, hyperpolarizing HCs from -14 to -54, -84, and -104 mV increased the amplitude of I(Ca) recorded from synaptically connected rods and caused hyperpolarizing shifts in I(Ca) voltage dependence. These effects were blocked by supplementing the bicarbonate-buffered saline solution with HEPES. In rods lacking light-responsive outer segments, hyperpolarizing neighboring HCs with light caused a negative activation shift and increased the amplitude of I(Ca). These changes in I(Ca) were blocked by HEPES and by inhibiting HC light responses with a glutamate antagonist, indicating that they were caused by HC feedback. These results show that rods, like cones, receive negative feedback from HCs that regulates the amplitude and voltage dependence of I(Ca). HC-to-rod feedback counters light-evoked decreases in synaptic output and thus shapes the transmission of rod responses to downstream visual neurons.

Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse.

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K(+) increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K(+)-induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.

Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro.

Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2/HCO3- buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2/HCO3- (5%/26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (fCN) recorded. Increases in fCN evoked by ACh, ATP and NaCN in CO2- free saline were significantly reduced (P < 0.05, Wilcoxon test) when CO2/HCO3- was present in the superfusion medium. Thus, the presence of CO2/HCO3- buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation.

Sustained in vivo activity of recombinant bovine granulocyte colony stimulating factor (rbG-CSF) using HEPES buffer.

The purpose of this study was to develop a long-acting injectable formulation of bG-CSF for veterinary use. However, in order to achieve sustained in vivo activity it was first necessary to stabilize the protein at the injection site. Preformulation studies, as well as literature, suggest that bG-CSF aggregates at neutral pH ranges (i.e., pH 6-8) and at temperatures of approximately 40 degrees C. Therefore, bG-CSF will not retain its activity for an extended period of time at the injection site. During this study we determined that HEPES buffer has a very significant impact on protein stability as well as on biological performance. Recombinant bovine granulocyte colony stimulating factor (rbG-CSF) was formulated in 1 M HEPES buffer for subcutaneous injection into cows. bG-CSF formulated in 1 M HEPES buffer resulted in sustained in vivo activity of bG-CSF compared to the "control" formulation (control formulation: 5% mannitol, 10 mM acetate buffer, 0.004% tween-80, pH 4). White blood cell (WBC) count was used as a marker to evaluate in vivo activity of the formulation. WBC numbers remained above a threshold value for only 24-30 h for the control formula. However, when bG-CSF was formulated in 1 M HEPES, the WBC remained above threshold for 3 days or 72 h. Formulating bG-CSF in 1 M HEPES at pH 7.5 also resulted in greater solution stability. This was surprising since bG-CSF is intrinsically not stable at neutral pH. The effect of 1 M HEPES on the T(M) (temperature at maximum heat flow on calorimetry scan) of bG-CSF was determined by microcalorimetry. In the absence of 1 M HEPES buffer the T(M) was 48 degrees C (onset approximately 40 degrees C), while bG-CSF formulated in 1 M HEPES buffer has a T(M) of 59 degrees C (onset approximately 50 degrees C). Similar organic buffers, such as MOPS, HEPPS, TES, and tricine, also resulted in improved solution stability as well as in sustained in vivo activity. The dramatic effect of these buffers on stability and biological performance of bG-CSF is not well understood. One hypothesis is that the electrostatic interaction between the zwitterionic form of these buffers and bG-CSF provides stabilization against denaturation.

The effect of intrinsic fluoride in cows' milk on in vitro enamel demineralization.

The fluoride concentration in cows' milk has been reported to vary with the fluoride levels in drinking water but it seldom exceeds 0.5 microg/ml. This raised a question as to whether any caries-protective effect could be attributed to the intrinsic fluoride of milk. Two samples of cows' milk with intrinsic fluoride concentrations of 0.03 and 0.3 microg/ml, respectively, were assessed for their protective effect on enamel in an in vitro demineralization model at relatively severe and mild acidic challenges (pH 4.6 and 5.0, respectively). Polished enamel discs were incubated individually in 5.0 ml of demineralization solution for 20 h per day alternated with 1-hour incubations in 1.0 ml of milk or control buffers: group 1, demineralization solution only (negative control); group 2, milk with 0.03 microg/ml fluoride; group 3, milk with 0.03 microg/ml fluoride; supplemented with NaF to 0.3 microg/ml fluoride; group 4, milk with 0.3 microg/ml fluoride; group 5, 0.3 microg/ml fluoride in 20 mM HEPES, pH 6.7; group 6, milk with 0.03 microg/ml fluoride supplemented with NaF to 5.0 microg/ml fluoride (positive control). The solutions were renewed each day and the calcium concentration in the demineralization solutions was followed during 4 days. The results showed that the protective effect of intrinsic milk fluoride on enamel is limited by the severity of the acidic challenge: There was a significant inhibition of the demineralization in groups 3-6 compared to groups 1 and 2, but only at pH 5.0 (p<0.0001) and not at pH 4.6 (p = 0.2). The organic components of milk had limited protection against demineralization because milk and HEPES with the same fluoride concentration gave similar results. The 36% reduction in calcium loss at pH 5.0 by treatment with milk with only 0.3 microg/ml fluoride is an indication that intrinsic milk fluoride has some caries-protective properties.

Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2.

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.

Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles.

We investigated the effects of reduced temperature, the pH elevators NH4Cl, monensin, and HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer, as well as the metabolic poisons NaF/KCN on transport of the fluid phase pinocytic marker, horseradish peroxidase (HRP), to lysosomes in Chinese hamster ovary (CHO) cells. In cell fractionation experiments, these agents appeared to block HRP transit at specific point(s) from "early" to "late" (i.e., low to high density) prelysosomal vesicles and lysosomes. Reduced temperature (17 degrees C) most strongly inhibited HRP transport from low density, early endosomes to lysosomes. In long-term HRP uptakes at 17 degrees C, marked peroxidase accumulation occurred both in early endosomes and in lysosomes. Loss (reversible pinocytosis) of HRP from "very early" endosomes occurred at 17 degrees C. All three pH elevators including the common media supplement HEPES buffer inhibited transit of internalized HRP into lysosomes. For all three pH elevators, inhibition was most pronounced at the "early" endosome stage. The respiratory inhibitors NaF/KCN also inhibited transport most strongly at the early endosome stage. Together these results suggest that "early" steps in the endocytic transport of HRP are the most sensitive and that the conditions tested may exert direct effects on the processing of endocytic vesicles.